SMART MODE:

NORMAL GENOMIC

• Jin T et al.
• Biological and genomic analysis of a PBSX-like defective phage induced from Bacillus pumilus AB94180.
• Arch Virol. 2014; 159: 739-52
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• Defective prophages, which are found in the genomes of many bacteria, are unable to complete a viral replication cycle and propagate in their hosts as healthy prophages. They package random DNA fragments derived from various sites of the host chromosome instead of their own genomes. In this study, we characterized a defective phage, PBP180, which was induced from Bacillus pumilus AB94180 by treatment with mitomycin C. Electron microscopy showed that the PBP180 particle has a head with a hexagonal outline of ~40 nm in diameter and a long tail. The DNA packaged in the PBP180 head consists of 8-kb DNA fragments from random portions of the host chromosome. The head and tail proteins of the PBP180 particle consist of four major proteins of approximately 49, 33, 16 and 14 kDa. The protein profile of PBP180 is different from that of PBSX, a well-known defective phage induced from Bacillus subtilis 168. A killing activity test against two susceptible strains each of B. subtilis and B. pumilus showed that the defective particles of PBP180 killed three strains other than its own host, B. pumilus AB94180, differing from the host-killing ranges of the defective phages PBSX, PBSZ (induced from B. subtilis W23), and PBSX4 (induced from B. pumilus AB94044). The genome of the PBP180 prophage, which is integrated in the B. pumilus AB94180 chromosome, is 28,205 bp in length, with 40 predicted open reading frames (ORFs). Further genomic comparison of prophages PBP180, PBSX, PBSZ and other PBSX-like prophage elements in B. pumilus strains revealed that their overall architectures are similar, but significant low homology exists in ORF29-ORF38, which presumably encode tail fiber proteins involved in recognition and killing of susceptible strains.

• Morcos F, Schafer NP, Cheng RR, Onuchic JN, Wolynes PG
• Coevolutionary information, protein folding landscapes, and the thermodynamics of natural selection.
• Proc Natl Acad Sci U S A. 2014; 111: 12408-13
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• The energy landscape used by nature over evolutionary timescales to select protein sequences is essentially the same as the one that folds these sequences into functioning proteins, sometimes in microseconds. We show that genomic data, physical coarse-grained free energy functions, and family-specific information theoretic models can be combined to give consistent estimates of energy landscape characteristics of natural proteins. One such characteristic is the effective temperature T(sel) at which these foldable sequences have been selected in sequence space by evolution. T(sel) quantifies the importance of folded-state energetics and structural specificity for molecular evolution. Across all protein families studied, our estimates for T(sel) are well below the experimental folding temperatures, indicating that the energy landscapes of natural foldable proteins are strongly funneled toward the native state.

• Milunovic B, diCenzo GC, Morton RA, Finan TM
• Cell growth inhibition upon deletion of four toxin-antitoxin loci from the megaplasmids of Sinorhizobium meliloti.
• J Bacteriol. 2014; 196: 811-24
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• Toxin and antitoxin (TA) gene pairs are addiction systems that are present in many microbial genomes. Sinorhizobium meliloti is an N2-fixing bacterial symbiont of alfalfa and other leguminous plants, and its genome consists of three large replicons, a circular chromosome (3.7 Mb) and the megaplasmids pSymA (1.4 Mb) and pSymB (1.7 Mb). S. meliloti carries 211 predicted type II TA genes, each encoding a toxin or an antitoxin. We constructed defined deletion strains that collectively removed the entire pSymA and pSymB megaplasmids except for their oriV regions. Of approximately 100 TA genes on pSymA and pSymB, we identified four whose loss was associated with cell death or stasis unless copies of the genes were supplied in trans. Orthologs of three of these loci have been characterized in other organisms (relB/E [sma0471/sma0473], Fic [DOC] [sma2105], and VapC [PIN] [orf2230/sma2231]), and this report contains the first experimental proof that RES/Xre (smb21127/smb21128) loci can function as a TA system. Transcriptome sequencing (RNA-seq) analysis did not reveal transcriptional differences between the TA systems to account for why deletion of the four "active" systems resulted in cell toxicity. These data suggest that severe cell growth phenotypes result from the loss of a few TA systems and that loss of most TA systems may result in more subtle phenotypes. These four TA systems do not appear to play a direct role in the S. meliloti-alfalfa symbiosis, as strains lacking these TA systems had a symbiotic N2 fixation phenotype that was indistinguishable from the wild type.

• Clifton MC et al.
• Structure of the Reston ebolavirus VP30 C-terminal domain.
• Acta Crystallogr F Struct Biol Commun. 2014; 70: 457-60
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• The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

• Perlinska A, Grynberg M
• Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin.
• PeerJ. 2014; 2: 679-679
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• Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche.

• Kimura T, Amaya Y, Kobayashi K, Ogasawara N, Sato T
• Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells.
• J Bacteriol. 2010; 192: 6209-16
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• The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.

• Reddy MC, Gokulan K, Jacobs WR Jr, Ioerger TR, Sacchettini JC
• Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation response.
• Protein Sci. 2008; 17: 159-70
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• The bacterial leucine-responsive regulatory protein (Lrp) is a global transcriptional regulator that controls the expression of many genes during starvation and the transition to stationary phase. The Mycobacterium tuberculosis gene Rv3291c encodes a 150-amino acid protein (designated here as Mtb LrpA) with homology with Escherichia coli Lrp. The crystal structure of the native form of Mtb LrpA was solved at 2.1 A. The Mtb LrpA structure shows an N-terminal DNA-binding domain with a helix-turn-helix (HTH) motif, and a C-terminal regulatory domain. In comparison to the complex of E. coli AsnC with asparagine, the effector-binding pocket (including loop 100-106) in LrpA appears to be largely preserved, with hydrophobic substitutions consistent with its specificity for leucine. The effector-binding pocket is formed at the interface between adjacent dimers, with an opening to the core of the octamer as in AsnC, and an additional substrate-access channel opening to the outer surface of the octamer. Using electrophoretic mobility shift assays, purified Mtb LrpA protein was shown to form a protein-DNA complex with the lat promoter, demonstrating that the lat operon is a direct target of LrpA. Using computational analysis, a putative motif is identified in this region that is also present upstream of other operons differentially regulated under starvation. This study provides insights into the potential role of LrpA as a global regulator in the transition of M. tuberculosis to a persistent state.

• Pedersen M, Lo Leggio L, Grossmann JG, Larsen S, Hammer K
• Identification of quaternary structure and functional domains of the CI repressor from bacteriophage TP901-1.
• J Mol Biol. 2008; 376: 983-96
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• The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein CI, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P(L) and the lysogenic promoter P(R) by binding to multiple operator sites on the DNA. In this study, we used a small bistable genetic switch element from phage TP901-1 to study the effect of cI deletions in vivo and showed that 43 amino acids could be removed from the C-terminal end of CI without destroying the ability of CI to repress transcription from the P(L) or the bistable switch properties. We showed that a helix-turn-helix motif located in the N-terminal part of CI is involved in DNA binding by introducing specific point mutations. Purification of CI and truncated forms of CI followed by analytical gel filtration and chemical cross-linking demonstrated that the C-terminal end of CI was required for oligomerization and that CI may exist as a hexamer in solution. Furthermore, expression and purification of the C-terminal part of CI (amino acids 92-180) showed that this part of the protein contained all the amino acids required to form an oligomer with an apparent molecular weight corresponding to a hexamer. We found that the C-terminal end of CI was required for de-repression of the P(L) following SOS induction, suggesting that the hexameric form of CI is needed for this or that this part of the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably a trimer of dimers.

• Miyakoshi M, Shintani M, Terabayashi T, Kai S, Yamane H, Nojiri H
• Transcriptome analysis of Pseudomonas putida KT2440 harboring the completely sequenced IncP-7 plasmid pCAR1.
• J Bacteriol. 2007; 189: 6849-60
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• The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal cat and pca genes involved in the catechol branch of the beta-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next, to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.

• Gorelik M, Lunin VV, Skarina T, Savchenko A
• Structural characterization of GntR/HutC family signaling domain.
• Protein Sci. 2006; 15: 1506-11
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• The crystal structure of Escherichia coli PhnF C-terminal domain (C-PhnF) was solved at 1.7 A resolution by the single wavelength anomalous dispersion (SAD) method. The PhnF protein belongs to the HutC subfamily of the large GntR transcriptional regulator family. Members of this family share similar N-terminal DNA-binding domains, but are divided into four subfamilies according to their heterogenic C-terminal domains, which are involved in effector binding and oligomerization. The C-PhnF structure provides for the first time the scaffold of this domain for the HutC subfamily, which covers about 31% of GntR-like regulators. The structure represents a mixture of alpha-helices and beta-strands, with a six-stranded antiparallel beta-sheet at the core. C-PhnF monomers form a dimer by establishing interdomain eight-strand beta-sheets that include core antiparallel and N-terminal two-strand parallel beta-sheets from each monomer. C-PhnF shares strong structural similarity with the chorismate lyase fold, which features a buried active site locked behind two helix-turn-helix loops. The structural comparison of the C-PhnF and UbiC proteins allows us to propose that a similar site in the PhnF structure is adapted for effector binding.

• Barragan MJ et al.
• BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators.
• J Biol Chem. 2005; 280: 10683-94
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• In this work, we have studied the transcriptional regulation of the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB. The transcription start site of the P(N) promoter running the expression of the bzd catabolic genes was identified. Gel retardation assays and P(N)::lacZ translational fusion experiments performed both in Azoarcus sp. CIB and Escherichia coli cells have shown that bzdR encodes a specific repressor that controls the inducible expression of the adjacent bzd catabolic operon, being the first intermediate of the catabolic pathway (i.e. benzoyl-CoA, the actual inducer molecule). This is the first report of a transcriptional repressor and a CoA-derived aromatic inducer controlling gene expression in the anaerobic catabolism of aromatic compounds. DNase I footprinting experiments revealed that BzdR protected three regions (operators) at the P(N) promoter. The three operators contain direct repetitions of a TGCA sequence that forms part of longer palindromic structures. In agreement with the repressor role of BzdR, operator region I spans the transcription initiation site as well as the -10 sequence for recognition of the RNA polymerase. Primary sequence analyses of BzdR showed an unusual modular organization with an N-terminal region homologous to members of the HTH-XRE family of transcriptional regulators and a C-terminal region similar to shikimate kinases. A three-dimensional model of the N-terminal and C-terminal regions of BzdR, generated by comparison with the crystal structures of the SinR regulator from Bacillus subtilis and the shikimate kinase I protein from E. coli, strongly suggests that they contain the helix-turn-helix DNA-binding motif and the benzoyl-CoA binding groove, respectively. The BzdR protein constitutes, therefore, the prototype of a new subfamily of transcriptional regulators.

• McLuskey K, Cameron S, Hammerschmidt F, Hunter WN
• Structure and reactivity of hydroxypropylphosphonic acid epoxidase in fosfomycin biosynthesis by a cation- and flavin-dependent mechanism.
• Proc Natl Acad Sci U S A. 2005; 102: 14221-6
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• The biosynthesis of fosfomycin, an oxirane antibiotic in clinical use, involves a unique epoxidation catalyzed by (S)-2-hydroxypropylphosphonic acid epoxidase (HPPE). The reaction is essentially dehydrogenation of a secondary alcohol. A high-resolution crystallographic analysis reveals that the HPPE subunit displays a two-domain combination. The C-terminal or catalytic domain has the cupin fold that binds a divalent cation, whereas the N-terminal domain carries a helix-turn-helix motif with putative DNA-binding helices positioned 34 A apart. The structure of HPPE serves as a model for numerous proteins, of ill-defined function, predicted to be transcription factors but carrying a cupin domain at the C terminus. Structure-reactivity analyses reveal conformational changes near the catalytic center driven by the presence or absence of ligand, that HPPE is a Zn(2+)/Fe(2+)-dependent epoxidase, proof that flavin mononucleotide is required for catalysis, and allow us to propose a simple mechanism that is compatible with previous experimental data. The participation of the redox inert Zn(2+) in the mechanism is surprising and indicates that Lewis acid properties of the metal ions are sufficient to polarize the substrate and, aided by flavin mononucleotide reduction, facilitate the epoxidation.

• Nelson D
• Phage taxonomy: we agree to disagree.
• J Bacteriol. 2004; 186: 7029-31

• Fukumori T, Morita Y, Tamiya E, Yokoyama K
• Design of peptide that recognizes double-stranded DNA.
• Anal Sci. 2003; 19: 181-3
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• A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.

• LeFevre KR, Cordes MH
• Retroevolution of lambda Cro toward a stable monomer.
• Proc Natl Acad Sci U S A. 2003; 100: 2345-50
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• The Cro protein from bacteriophage lambda has a dimeric alpha+beta fold that evolved from an ancestral all-alpha monomer. The sequence mutations responsible for this dramatic structural evolution are unknown. Here we use analysis of sequence alignments to show that Ala-33, a small side chain in the hydrophobic "ball-and-socket" dimer interface of lambda Cro, was a much larger tryptophan side chain at a previous point in evolution. The retroevolutionary lambda Cro-A33W mutant shows a 10-fold reduction in dimerization affinity relative to the wild type as well as a large increase in monomer thermal stability (Delta T(m) > 10 degrees C), apparently due to partial filling of the hydrophobic socket from within the same monomer. An additional mutation in the dimer interface, F58D, almost completely abolishes detectable dimerization while maintaining the high monomer stability. The secondary structure content of the monomerized versions of lambda Cro is similar to that of the wild-type protein, and the tertiary structure of the monomer appears relatively well defined. These results (i) support a model in which the ball-and-socket dimer interface of lambda Cro was created by altered volume mutations within a limited branch of the Cro lineage and (ii) suggest the possibility that the evolution of the alpha+beta dimer from an all-alpha monomer proceeded through an alpha+beta monomer intermediate.

• Zaim J, Kierzek AM
• The structure of full-length LysR-type transcriptional regulators. Modeling of the full-length OxyR transcription factor dimer.
• Nucleic Acids Res. 2003; 31: 1444-54
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• The LysR-type transcriptional regulators (LTTRs) comprise the largest family of prokaryotic transcription factors. These proteins are composed of an N-terminal DNA binding domain (DBD) and a C-terminal cofactor binding domain. To date, no structure of the DBD has been solved. According to the SUPERFAMILY and MODBASE databases, a reliable homology model of LTTR DBDs may be built using the structure of the Escherichia coli ModE transcription factor, containing a winged helix- turn-helix (HTH) motif, as a template. The remote, but statistically significant, sequence similarity between ModE and LTTR DBDs and an alignment generated using SUPERFAMILY and MODBASE methods was independently confirmed by alignment of sequence profiles representing ModE and LTTR family DBDs. Using the crystal structure of the E.coli OxyR C-terminal domain and the DBD alignments we constructed a structural model of the full-length dimer of this LTTR family member and used it to investigate the mode of protein-DNA interaction. We also applied the model to interpret, in a structural context, the results of numerous biochemical studies of mutated LTTRs. A comparison of the LTTR DBD model with the structures of other HTH proteins also provides insights into the interaction of LTTRs with the C-terminal domain of the RNA polymerase alpha subunit.

• Binishofer B, Moll I, Henrich B, Blasi U
• Inducible promoter-repressor system from the Lactobacillus casei phage phiFSW.
• Appl Environ Microbiol. 2002; 68: 4132-5
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• With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage phiFSW-TI in Escherichia coli. The phiFSW-TI promoter fragment was abutted to the plasmid-borne promoterless beta-glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei phiFSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated phiFSW-TI promoter, was repressed at 28 degrees C and expressed at 42 degrees C. Moreover, a homology search revealed that the C terminus of the isolated phiFSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.

• Salmon KA, Freedman O, Ritchings BW, DuBow MS
• Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112.
• Virology. 2000; 272: 85-97
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• Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.

• Zinser ER, Kolter R
• Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.
• J Bacteriol. 2000; 182: 4361-5
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• Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almiron, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of an rpoS strain had acquired a GASP mutation in lrp. This GASP allele, lrp-1141, encodes a mutant protein lacking the critical glycine in the turn of the helix-turn-helix DNA-binding domain. The lrp-1141 allele behaves as a null mutation when in single copy and is dominant negative when overexpressed. Hence, the mutant protein appears to retain stability and the ability to dimerize but lacks DNA-binding activity. We also demonstrated that a lrp null allele generated by a transposon insertion has a fitness gain identical to that of the lrp-1141 allele, verifying that cells lacking Lrp activity have a competitive advantage during prolonged starvation. Finally, we tested by genetic analysis the hypothesis that the lrp-1141 GASP mutation confers a fitness gain by enhancing amino acid catabolism during carbon starvation. We found that while amino acid catabolism may play a role, it is not necessary for the lrp GASP phenotype, and hence the lrp GASP phenotype is due to more global physiological changes.

• Ladero V, Garcia P, Bascaran V, Herrero M, Alvarez MA, Suarez JE
• Identification of the repressor-encoding gene of the Lactobacillus bacteriophage A2.
• J Bacteriol. 1998; 180: 3474-6
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• The repressor gene of the Lactobacillus phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the lambda Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected.

• Cook WJ, Kar SR, Taylor KB, Hall LM
• Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins.
• J Mol Biol. 1998; 275: 337-46
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• SmtB from Synechococcus PCC7942 is a trans-acting dimeric repressor that is required for Zn(2+)-responsive expression of the metallothionein SmtA. The structure of SmtB was solved using multiple isomorphous replacement techniques and refined at 2.2 A resolution by simulated annealing to an R-factor of 0.218. SmtB displays the classical helix-turn-helix motif found in many DNA-binding proteins. It has an alpha + beta topology, and the arrangement of the three core helices and the beta hairpin is similar to the HNF-3/fork head, CAP and diphtheria toxin repressor proteins. Although there is no zinc in the crystal structure, analysis of a mercuric acetate derivative suggests a total of four Zn2+ binding sites in the dimer. Two of these putative sites are at the opposite ends of the dimer, while the other two are at the dimer interface and are formed by residues contributed from each monomer. The structure of the dimer is such that simultaneous binding for both recognition helices to DNA would require either a bend in the DNA helix or a conformational change in the dimer. The structure of Synechococcus SmtB is the first in this family of metal-binding DNA repressors.

• Alexandrov NN, Luethy R
• Alignment algorithm for homology modeling and threading.
• Protein Sci. 1998; 7: 254-8
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• A DNA/protein sequence comparison is a popular computational tool for molecular biologists. Finding a good alignment implies an evolutionary and/or functional relationship between proteins or genomic loci. Sequential similarity between two proteins indicates their structural resemblance, providing a practical approach for structural modeling, when structure of one of these proteins is known. The first step in the homology modeling is a construction of an accurate sequence alignment. The commonly used alignment algorithms do not provide an adequate treatment of the structurally mismatched residues in locally dissimilar regions. We propose a simple modification of the existing alignment algorithm which treats these regions properly and demonstrate how this modification improves sequence alignments in real proteins.

• Narumi I, Cherdchu K, Kitayama S, Watanabe H
• The Deinococcus radiodurans uvr A gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria.
• Gene. 1997; 198: 115-26
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• Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

• Backes H, Berens C, Helbl V, Walter S, Schmid FX, Hillen W
• Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation.
• Biochemistry. 1997; 36: 5311-22
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• We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.

• Bruttin A, Desiere F, Lucchini S, Foley S, Brussow H
• Characterization of the lysogeny DNA module from the temperate Streptococcus thermophilus bacteriophage phi Sfi21.
• Virology. 1997; 233: 136-48
• Display abstract

• Phage phi Sfi21, the only temperate Streptococcus thermophilus phage from our phage collection, showed extensive DNA homology with virulent phages from lytic group I. Southern blot hybridizations demonstrated that the phi Sfi21-specific DNA was clustered in an approximately 6.6-kb-long region, the putative lysogeny module. Sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage BK5-T; orf 127 and orf 122 with weak homology to the N- and C-terminal parts, respectively, of the cl-like repressor from lactococcal phages Tuc2009 and BK5-T; orf 75 with homology to a repressor protein from lambdoid phage 434 and an anti-repressor ant with homology to phage P1. The molecular arrangement of the predicted orfs in phage phi Sfi21 was very similar to that of the lactococcal phage BK5-T. The transition from phi Sfi21-specific DNA into DNA shared with virulent phages was abrupt and flanked at one side by notable DNA repeats. Sequence analysis identified a holin protein to the left of the lysogeny module. A site-specific deletion of 2.4 kb, which reproducibly transformed phi Sfi21 into a lytic phage, was localized in the lysogeny module. It was flanked at both sides by conspicuous DNA repeats. One repeat region reflected the DNA around the attP site, while the other reflected the putative genetic switch region between repressor and anti-repressor genes. S. thermophilus host Sfi1 transformed with a plasmid containing int and orf 203 showed resistance to superinfection by heterologous phages, but not by the homologous phi Sfi21. Part of the int gene could be deleted without loss of this activity, while a deletion in orf 203 resulted in loss of the phage resistance. We speculate on the possibility of a bipartite immunity system for the control of lysogeny in phi Sfi21.

• Zeng G, Ye S, Larson TJ
• Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identification of the DNA-binding domain.
• J Bacteriol. 1996; 178: 7080-9
• Display abstract

• The nucleotide sequence of the glpEGR operon of Escherichia coli was determined. The translational reading frame at the beginning, middle, and end of each gene was verified. The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082. The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278. The functions of GlpE and GlpG are unknown. The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal. The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding. Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro. However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors. For the first time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcriptional regulators in the deoR family.

• Saxild HH, Andersen LN, Hammer K
• Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.
• J Bacteriol. 1996; 178: 424-34
• Display abstract

• The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

• Marasco R, Varcamonti M, Ricca E, Sacco M
• A new Bacillus subtilis gene with homology to Escherichia coli prc.
• Gene. 1996; 183: 149-52
• Display abstract

• We report the cloning of a 2-kb PstI-BamHI fragment of Bacillus subtilis DNA carrying an open reading frame of 1398 bp, herein designated orfRM1. This orf was shown to be transcribed only during vegetative growth from a putative sigma A-specific promoter. The deduced amino acid sequence predicted a polypeptide of 51 kDa (466 aa), which shows significant percentage of identity with the Escherichia coli Prc protein. However no Prc-like phenotypes were observed in a B. subtilis orfRM1 deletion-insertion mutant.

• Gering M, Bruckner R
• Transcriptional regulation of the sucrase gene of Staphylococcus xylosus by the repressor ScrR.
• J Bacteriol. 1996; 178: 462-9
• Display abstract

• In Staphylococcus xylosus, scrB is one of two genes necessary for sucrose utilization. It encodes a sucrase that hydrolyzes intracellular sucrose-6-phosphate generated by the uptake of sucrose via the sucrose-specific enzyme II of the phosphotransferase system, the gene product of scrA. ScrB sucrase activity is inducible by the presence of sucrose in the culture medium. Primer extension experiments demonstrated that the observed regulation is achieved at the level of scrB transcription initiation. The protein mediating sucrose-specific regulation of scrB was found to be encoded immediately upstream of the sucrase gene. The nucleotide sequence of the regulatory gene scrR comprises an open reading frame that specifies a protein of 35.8 kDa. This protein exhibits similarity to transcriptional regulators of the GalR-LacI family. Inactivation of the scrR reading frame in the genome of S. xylosus led to the constitutive expression of scrB at a high level, identifying ScrR as a repressor of transcription. Sucrose-specific regulation of scrB was also lost upon deletion of 4 bp of a palindromic sequence (OB) covering positions +6 to +21 downstream of the scrB transcriptional start site. These results suggested a direct interaction of the ScrR repressor and the operator OB. Accordingly, a fusion protein consisting of the maltose-binding protein of Escherichia coli and the ScrR protein was able to interact with an scrB promoter fragment in gel mobility shift experiments but failed to bind an scrB fragment carrying the 4-bp deletion derivative of OB. An scrR promoter fragment, which dose not contain a sequence resembling OB, was not shifted by the fusion protein. This result corroborates scrR primer extension analyses showing that transcription of the repressor gene itself is not regulated. Therefore, the sucrase gene operator OB is the target sequence through which the ScrR protein exerts its negative effect on transcription initiation. In the promoter region of scrA, the gene essential for sucrose transport, two palindromic sequences that are similar to the scrB operator are found. Their presence in scrA suggests that ScrR controls a sucrose-specific regulon in S. xylosus.

• Li N, Chen Y, Feng J
• [Cloning and analysis of prophage PBSX repressor gene from Bacillus subtilis].
• Yi Chuan Xue Bao. 1995; 22: 478-86
• Display abstract

• The repressor gene and its allele--a temperature sensitive mutant have been cloned from the defective prophage PBSX of Bacillus subtilis by means of PCR technique. The characterization of these sequences suggest that wild-type and mutant gene have the same open reading frame, which probably encode a repressor protein of 113 amino acids, and the putative promoter region and ribosome binding sites within the sequences have also been identified. The results from complementation experiments demonstrate that repressor encoded by wild-type gene is capable of blocking the temperature-shift to induce PBSX prophage in Bacillus subtilis.

• Kobayashi K, Shoji K, Shimizu T, Nakano K, Sato T, Kobayashi Y
• Analysis of a suppressor mutation ssb (kinC) of sur0B20 (spo0A) mutation in Bacillus subtilis reveals that kinC encodes a histidine protein kinase.
• J Bacteriol. 1995; 177: 176-82
• Display abstract

• sur0B20 is a mutation that suppresses the effects of spo0B delta B or spo0F221 mutations in Bacillus subtilis, sur0B20 is an allele of the spo0A gene (Glu-14 to Val-14 conversion) and restores the sporulation of spo0B or spo0F mutants to the wild-type level. Here, we report the isolation of suppressor mutations of sur0B20 (ssb). One of these mutations, ssb-12, severely impairs the suppressor activity of sur0B20. A 2.5-kbp MboI fragment which complements the ssb-12 mutation was cloned by the prophage transformation method using phi CM as a vector. Nucleotide sequencing of the fragment revealed two open reading frames (orf1 and orf2). Gene disruption and complementation experiments showed that orf2 is the ssb gene. ssb was shown to encode a protein with a molecular weight of 48,846 (428 amino acid residues) showing strong similarity to transmitter kinases, especially KinA, of two-component regulatory systems. Therefore, ssb was renamed kinC. Deletion of kinC had no observable effect on sporulation. kinC transcription was induced at the onset of sporulation, probably from a sigma A-dependent promoter, and its expression was shut off at T3. DNase I protection experiments showed that the Spo0A protein binds to two adjoining sites in the kinC promoter region with different affinities. These results suggest that kinC expression might be regulated by Spo0A.

• Jancso A, Botfield MC, Sowers LC, Weiss MA
• An altered-specificity mutation in a human POU domain demonstrates functional analogy between the POU-specific subdomain and phage lambda repressor.
• Proc Natl Acad Sci U S A. 1994; 91: 3887-91
• Display abstract

• The POU motif, conserved among a family of eukaryotic transcription factors, contains two DNA-binding domains: an N-terminal POU-specific domain (POUS) and a C-terminal homeodomain (POUHD). Surprisingly, POUS is similar in structure to the helix-turn-helix domains of bacteriophage repressor and Cro proteins. Such similarity predicts a common mechanism of DNA recognition. To test this prediction, we have studied the DNA-binding properties of the human Oct-2 POU domain by combined application of chemical synthesis and site-directed mutagenesis. The POUS footprint of DNA contacts, identified by use of modified bases, is analogous to those of bacteriophage repressor-operator complexes. Moreover, a loss-of-contact substitution in the putative POUS recognition alpha-helix leads to relaxed specificity at one position in the DNA target site. The implied side chain-base contact is identical to that of bacteriophage repressor and Cro proteins. These results establish a functional analogy between the POUS and prokaryotic helix-turn-helix elements and suggest that their DNA specificities may be governed by a shared set of rules.

• McDonnell GE, McConnell DJ
• Overproduction, isolation, and DNA-binding characteristics of Xre, the repressor protein from the Bacillus subtilis defective prophage PBSX.
• J Bacteriol. 1994; 176: 5831-4
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• PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Lysogeny is maintained by the PBSX-encoded repressor, Xre. The Xre protein was overproduced in Escherichia coli and isolated by affinity chromatography. Gel retardation and DNase I footprinting studies indicated that Xre binds to four sites close to its own gene. These sites overlap putative promoters for xre and a divergent transcriptional unit, containing the middle genes.

• Bowie JU, Eisenberg D
• An evolutionary approach to folding small alpha-helical proteins that uses sequence information and an empirical guiding fitness function.
• Proc Natl Acad Sci U S A. 1994; 91: 4436-40
• Display abstract

• Three short protein sequences have been guided by computer to folds resembling their crystal structures. Initially, peptide fragment conformations ranging in size from 9 to 25 residues were selected from a database of known protein structures. A fragment was selected if it was compatible with a segment of the sequence to be folded, as judged by three-dimensional profile scores. By linking the selected fragment conformations together, hundreds of trial structures were generated of the same length and sequence as the protein to be folded. These starting trial structures were then improved by an evolutionary algorithm. Selection pressure for improving the structures was provided by an energy function that was designed to guide the conformational search procedure toward the correct structure. We find that by evolution of only 400 structures for fewer than 1400 generations, the overall fold of some small helical proteins can be computed from the sequence, with deviations from observed structures of 2.5-4.0 A for C alpha atoms.

• Hua J, Patton JT
• The carboxyl-half of the rotavirus nonstructural protein NS53 (NSP1) is not required for virus replication.
• Virology. 1994; 198: 567-76
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• The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.

• Benevides JM, Weiss MA, Thomas GJ Jr
• An altered specificity mutation in the lambda repressor induces global reorganization of the protein-DNA interface.
• J Biol Chem. 1994; 269: 10869-78
• Display abstract

• The lambda repressor exhibits structural characteristics of lock and key complementary through the helix-turn-helix motif, and of induced fit by virtue of DNA-dependent folding of the N-terminal arm. In both cases, molecular recognition is mediated by direct contacts between amino acids and DNA bases. The extent to which such contacts function as discrete elements in a protein-DNA recognition code is not known. Because of the relevance of protein recognition to the broader issue of protein design, and because the lambda system serves as a prototype for gene regulation, we have employed laser Raman and 1H NMR spectroscopy to compare free and operator-bound structures of lambda repressor variants which are known to exhibit altered DNA-binding specificities. Experimental design is based upon a previous biochemical study of mutations in the repressor N-terminal arm (K4Q) and helix-turn-helix motif (G48S) (Nelson, H. C. M., and Sauer, R. T. (1986) J. Mol. Biol. 192, 27-38). These mutations, which were originally isolated by loss of function (K4Q) and second-site reversion (G48S), are of particular interest in light of their complex effects on sequence specificity at multiple positions in the operator site (Benson, N., Adams, C., and Youderian, P. (1992) Genetics 130, 17-26). Laser Raman and 1H NMR spectra of repressor variants carrying one (G48S) or two mutations (K4Q/G48S) are similar to those of the native wild type repressor and are in accord with the x-ray crystal structure. Remarkably, however, the complexes of wild type and mutant repressors exhibit extensive differences both in the global DNA structure and in the environments of key functional groups along the major groove. By demonstrating that single amino acid substitutions can induce global reorganization of a protein-DNA interface, the present results establish that repressor-operator recognition in solution cannot be explained in terms of a simple recognition code.

• Cohen SP, Hachler H, Levy SB
• Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli.
• J Bacteriol. 1993; 175: 1484-92
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• A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

• Goldstein RA, Luthey-Schulten ZA, Wolynes PG
• Optimal protein-folding codes from spin-glass theory.
• Proc Natl Acad Sci U S A. 1992; 89: 4918-22
• Display abstract

• Protein-folding codes embodied in sequence-dependent energy functions can be optimized using spin-glass theory. Optimal folding codes for associative-memory Hamiltonians based on aligned sequences are deduced. A screening method based on these codes correctly recognizes protein structures in the "twilight zone" of sequence identity in the overwhelming majority of cases. Simulated annealing for the optimally encoded Hamiltonian generally leads to qualitatively correct structures.

• Bowie JU, Sauer RT
• TraY proteins of F and related episomes are members of the Arc and Mnt repressor family.
• J Mol Biol. 1990; 211: 5-6
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• Amino acid changes known to be structurally allowed in Arc repressor were used to aid in the identification of proteins with sequence similarity to Arc and the related Mnt repressor. The sequences of the TraY proteins from the F episome and related episomes were found to be similar to those of Arc and Mnt.

• Pace HC, Lu P, Lewis M
• lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA.
• Proc Natl Acad Sci U S A. 1990; 87: 1870-3
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• The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer. The crystals without DNA diffract to better than 3.5 A. They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 A, b = 75.6 A, and c = 161.2 A, with alpha = gamma = 90 degrees and beta = 125.5 degrees. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 A. These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl beta-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.

• Bowie JU, Sauer RT
• Identifying determinants of folding and activity for a protein of unknown structure.
• Proc Natl Acad Sci U S A. 1989; 86: 2152-6
• Display abstract

• We have generated an extensive genetic map of functionally allowed and/or structurally allowed amino acid substitutions in Arc repressor, a DNA binding protein of unknown structure. Analysis of the allowed substitution patterns identifies residues that are likely to be involved in protein function and identifies side chains that play important structural roles, including residues likely to form the hydrophobic core. The identities of approximately one-third of the residues in Arc repressor are functionally important, about one-half are structurally important, and the remainder are unimportant for either structure or function. The patterns of obligatory hydrophobic positions permit strong predictions of secondary structure.

• Pakula AA, Sauer RT
• Amino acid substitutions that increase the thermal stability of the lambda Cro protein.
• Proteins. 1989; 5: 202-10
• Display abstract

• A mutant Cro protein, which bears the Ile-30----Leu substitution, is thermally unstable and degraded more rapidly than wild-type Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12 degrees C to 14 degrees C. These amino acid substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild-type Cro.

• Zaballos A, Lazaro JM, Mendez E, Mellado RP, Salas M
• Effects of internal deletions on the priming activity of the phage phi 29 terminal protein.
• Gene. 1989; 83: 187-95
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• A series of internal deletions of gene 3, coding for the phage phi 29 DNA terminal protein, have been constructed and characterized. In addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained. The priming activity of the substitution mutant protein, in the formation of the protein p3-dAMP initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function. Deletions of 8 to 33 aa, from aa residue 48 towards the N terminus of the substitution mutant, further decreased the priming activity of the protein. The activity of deletion mutants lacking 15 or 21 aa from residue 57 towards the C terminus, and also containing a point mutation at position 56, was greatly reduced, and no activity was seen when 24 aa were lacking.

• Breyer RM, Sauer RT
• Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor.
• J Biol Chem. 1989; 264: 13355-60
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• Monoclonal antibody 51F recognizes determinants in the helix 4 region of the native form of the N-terminal domain of lambda repressor. A cassette mutagenesis method was used to introduce changes within this region, and antibody-reactive candidates were isolated and sequenced. The resulting data allow the identification of repressor side chains that are critical determinants of antibody binding. Four of these side chains are on the surface of the N-terminal domain and probably contact the antibody directly. These contact positions were then mutagenized individually, and the antibody binding phenotypes of a large number of singly mutant repressors were determined. Taken together, the mutational data allow a functional map of the recognition surface to be constructed and the physical nature of some of the specific interactions that stabilize the antibody-antigen complex to be surmised.

• Bushman FD, Ptashne M
• Turning lambda Cro into a transcriptional activator.
• Cell. 1988; 54: 191-7
• Display abstract

• According to our present understanding, lambda repressor bound to DNA stimulates transcription by touching RNA polymerase bound at an adjacent promoter. The part of repressor required for activation was identified in part by the isolation of mutants specifically impaired in transcriptional activation. The amino acids of repressor altered in these "positive control" mutants lie in an acidic patch on the surface of repressor that is closely apposed to RNA polymerase. In this study, we show that this "activating patch" of repressor is sufficient for transcriptional activation in another sequence context. We transfer this activating patch onto the surface of lambda Cro, a protein normally unable to activate transcription, and show that the modified Cro is a transcriptional activator. In addition, we provide evidence that the repressor protein of phage 434 also activates transcription using an activating patch similar to that of lambda repressor.

• Sinclair RB, Bibb MJ
• The repressor gene (c) of the Streptomyces temperate phage phi c31: nucleotide sequence, analysis and functional cloning.
• Mol Gen Genet. 1988; 213: 269-77
• Display abstract

• The nucleotide sequence of the 3.4 kb SphI-G fragment that contained the repressor gene (c) of the temperate Streptomyces phage phi c31 was determined. Analysis of this sequence revealed a large open reading frame with protein coding character and sequence changes in c gene point and deletion mutants identified this as the coding region of the repressor. Two of the mutants studied had undergone deletions of 1.1 kb and 1.4 kb that had occurred across short direct repeats of 6 bp and 11 bp, respectively. Coupled in vitro transcription-translation experiments using the cloned SphI-G fragment and Streptomyces lividans cell free extracts identified a protein product of approximately 72 kDa, in close agreement with that predicted from the nucleotide sequence. A strongly predicted helix-turn-helix motif that may be involved in DNA binding occurred towards the carboxy-terminus of the amino acid sequence. Initial attempts to clone the SphI-G fragment in Streptomyces failed; using information gained from the sequence analysis a smaller segment of this DNA fragment was cloned in S. lividans and conferred immunity to a clear plaque mutant (c1) of phi c31.

• Oda M, Sugishita A, Furukawa K
• Cloning and nucleotide sequences of histidase and regulatory genes in the Bacillus subtilis hut operon and positive regulation of the operon.
• J Bacteriol. 1988; 170: 3199-205
• Display abstract

• An 8-kilobase HindIII fragment carrying the histidase gene (hutH) and its regulatory region (hutP), from the Bacillus subtilis histidine utilization (hut) operon, was cloned in the temperate bacteriophage phi 105. Histidine utilization was restored in a hutH1 mutant by the specialized transducing phage (phi 105hutH11). The histidase gene in phi 105hutH11 was inducible and was shown to be under catabolite repression. The nucleotide sequence of 3,932 base pairs including the hutH and hutP loci revealed three open reading frames (ORFs). The molecular weights of ORF1 and ORF2 proteins were calculated to be 16,576 (151 amino acid residues) and 55,675 (508 amino acid residues), respectively. Reverse transcriptase mapping experiments showed that the putative promoter for the hut operon could be recognized by RNA polymerase sigma 43. The transcript starts at an adenosine residue 32 base pairs upstream from the initiation codon of ORF1. hutH+-transforming activity was found in ORF2, indicating that ORF2 encoded the histidase. A hutP1 mutation was determined as a substitution of an amino acid in ORF1. By using a specialized transducing phage containing the wild-type ORF1 gene, it was demonstrated that the presence of ORF1 protein in trans was absolutely required for the induction of the hut operon in a hutP1 mutant. These data strongly suggested that ORF1 encodes a positive regulator of the hut operon.

• Weiss MA, Karplus M, Sauer RT
• Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins.
• J Biomol Struct Dyn. 1987; 5: 539-56
• Display abstract

• The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.

• Hogrefe HH, Griffith JP, Rossmann MG, Goldberg E
• Characterization of the antigenic sites on the refined 3-A resolution structure of mouse testicular lactate dehydrogenase C4.
• J Biol Chem. 1987; 262: 13155-62
• Display abstract

• The atomic structure of mouse testicular apolactate dehydrogenase C4 has been refined to 3.0-A resolution yielding a final crystallographic R-factor of 0.256. Comparison with the refined structure of dogfish apolactate dehydrogenase A4 shows that equivalent secondary structure elements are essentially in the same position relative to the molecular 2-fold axes, except for the helices alpha D, alpha E, and alpha 2G in the vicinity of the active center, and the carboxyl-terminal helix alpha H. The positions of antigenic peptides correlate best with surface accessibilities of the monomer rather than of the full tetrameric molecule.

• Vershon AK, Bowie JU, Karplus TM, Sauer RT
• Isolation and analysis of arc repressor mutants: evidence for an unusual mechanism of DNA binding.
• Proteins. 1986; 1: 302-11
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• We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.

• McLaughlin JR, Wong HC, Ting YE, Van Arsdell JN, Chang S
• Control of lysogeny and immunity of Bacillus subtilis temperate bacteriophage SP beta by its d gene.
• J Bacteriol. 1986; 167: 952-9
• Display abstract

• The d gene from the Bacillus subtilis temperate bacteriophage SP beta was isolated. When introduced into an SP beta-sensitive strain of B. subtilis, the cloned d gene directed the synthesis of a 22-kilodalton protein and conferred on the host immunity to SP beta phage. A frameshift mutation, designated d2, was introduced into the cloned d gene, and it was subsequently crossed back into the SP beta phage genome. The resulting SP beta phage grew lytically and formed clear plaques on sensitive bacteria. Although the cloned d gene confers immunity to the host, we could not detect complementation of the d gene by mixed infection with SP beta d2 and various SP beta c mutants. The nucleotide sequence of the 1,033-base-pair PstI-to-EcoRI fragment containing the d gene was determined; it includes an open reading frame that could potentially encode a protein of 227 amino acids. The gene was mapped within the PstI H fragment on the phage genome, which positions the d gene about 25 kilobases from the right end of the phage genome. It is transcribed from right to left.

• Eisenbeis SJ, Nasoff MS, Noble SA, Bracco LP, Dodds DR, Caruthers MH
• Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.
• Proc Natl Acad Sci U S A. 1985; 82: 1084-8
• Display abstract

• A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.

• Isackson PJ, Bertrand KP
• Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding.
• Proc Natl Acad Sci U S A. 1985; 82: 6226-30
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• The Tn10 tet repressor regulates transcription of the tetracycline-resistance determinant in transposon Tn10. Previous DNA sequencing studies identified a region of tet repressor (amino acids 26-47) that is homologous to the helix-turn-helix regions of lambda Cro, lambda repressor, and catabolite gene activator protein that are implicated in sequence-specific DNA binding. Here we report the isolation of dominant tetR mutations that result in tet repressors deficient in tet operator binding but that retain some capacity to form dimers with, and thereby inactivate, wild-type repressor monomers. The mutations were isolated by transforming a tetR+ tetA-lacZ fusion strain with hydroxylamine-mutagenized tetR plasmid DNA and then screening for increased lacZ expression. DNA sequence analysis of 35 independent isolates identified seven different mutations, five of which are in the region of helix-turn-helix sequence homology. In vitro binding studies indicate that the mutations in this region of tet repressor reduce the affinity of tet repressor for tet operator DNA by at least a factor of 1000 but have no significant effect on the affinity of tet repressor for tetracycline. These results provide strong support for the proposal that tet repressor utilizes the conserved helix-turn-helix structural motif in binding to tet operator DNA.

• Ohlendorf DH, Anderson WF, Matthews BW
• Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor.
• J Mol Evol. 1983; 19: 109-14
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• Amino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common an homologous region of about 22 amino acids. This region corresponds to two consecutive alpha-helices that occur in both Cro and cI repressor proteins of bacteriophage lambda and in catabolite gene activator protein of Escherichia coli and are presumed to interact with DNA. The results obtained here suggest that this alpha-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.

• Ohlendorf DH, Anderson WF, Fisher RG, Takeda Y, Matthews BW
• The molecular basis of DNA-protein recognition inferred from the structure of cro repressor.
• Nature. 1982; 298: 718-23
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• Recognition by cro repressor protein of its specific DNA binding sites appears to occur via multidentate hydrogen bonds between amino acid side chains of the protein and base-pair atoms in the major groove of right-handed B-form DNA. Most of the sequence-specific interactions between cro and DNA, as well as a number of sequence-independent ones, are mediated by a two-alpha-helical unit which appears to be common to many proteins that regulate gene expression.

• Anderson WF, Ohlendorf DH, Takeda Y, Matthews BW
• Structure of the cro repressor from bacteriophage lambda and its interaction with DNA.
• Nature. 1981; 290: 754-8
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• The three-dimensional structure of the 66-amino acid cro repressor protein of bacteriophage lambda suggests how it binds to its operator DNA. We propose that a dimer of cro protein is bound to the B-form of DNA with the 2-fold axis of the dimer coincident with the 2-fold axis of DNA. A pair of 2-fold-related alpha-helices of the repressor, lying within successive major grooves of the DNA, seem to be a major determinant in recognition and binding. In addition, the C-terminal residues of the protein, some of which are disordered in the absence of DNA, appear to contribute to the binding.

• Hunt S, Fraser RD, MacRae TP, Suzuki E
• Molecular organization in molluscan operculae.
• J Mol Biol. 1979; 129: 149-53

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