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S W White

Publications and source records attributed to S W White.

At least 19 recordsLinked to original sources
PubMed1998

The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif.

We report the 1.7 A crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Delta41). S4Delta41 has two domains; domain 1 is completely alpha-helical and domain 2 comprises a five-stranded antiparallel beta-sheet with three alpha-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1998

Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance.

Antibiotic resistance is rapidly becoming a major medical problem. Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective. It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance. Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome. Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations. The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin. The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin. In both proteins, the mutations occur within their putative RNA-binding sites. The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region. These results support the hypothesis that the mutations create local distortions of the catalytic RNA component.When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery. We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic. As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu.GTP.aminoacyl tRNA binding site on the large subunit.

Amino Acid Sequence↗
PubMed1998

Conformational variability of the N-terminal helix in the structure of ribosomal protein S15.

BACKGROUND: Ribosomal protein S15 is a primary RNA-binding protein that binds to the central domain of 16S rRNA. S15 also regulates its own synthesis by binding to its own mRNA. The binding sites for S15 on both mRNA and rRNA have been narrowed down to less than a hundred nucleotides each, making the protein an attractive candidate for the study of protein-RNA interactions. RESULTS: The crystal structure of S15 from Bacillus stearothermophilus has been solved to 2.1 A resolution. The structure consists of four alpha helices. Three of these helices form the core of the protein, while the N-terminal helix protrudes out from the body of the molecule to make contacts with a neighboring molecule in the crystal lattice. S15 contains a large conserved patch of basic residues which could provide a site for binding 16S rRNA. CONCLUSIONS: The conformation of the N-terminal alpha helix is quite different from that reported in a recent NMR structure of S15 from Thermus thermophilus. The intermolecular contacts that this alpha helix makes with a neighboring molecule in the crystal, however, closely resemble the intramolecular contacts that occur in the NMR structure. This conformational variability of the N-terminal helix has implications for the range of possible S15-RNA interactions. A large, conserved basic patch at one end of S15 and a cluster of conserved but exposed aromatic residues at the other end provide two possible RNA-binding sites on S15.

Amino Acid Sequence↗
PubMed1998

Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome.

Models of the bacterial ribosome based on recent structural analyses are beginning to provide new insights into the protein synthetic machinery. Central to evolving models are the high-resolution structures of individual ribosomal proteins, which represent detailed probes of their local RNA and protein environments. Ribosomal proteins are extremely ancient molecules; the structures therefore also provide a unique window into early protein evolution. Many of the proteins contain domains that are present in more recently evolved families of RNA- and DNA-binding proteins. Such structural homology can be used to predict mechanisms by which proteins interact with RNA in the ribosome.

Bacteria↗
PubMed1998

Evidence of baroreflex adaptation to repetitive +Gz in fighter pilots.

BACKGROUND: The arterial baroreflex acts to maintain arterial pressure in the face of an orthostatic challenge. The high +Gz loads experienced by fighter pilots represent an extreme form of orthostatic challenge. G-induced loss of consciousness (G-LOC) represents a failure of the baroreflex system to maintain the appropriate level of cerebral perfusion. The anecdotal experience of fighter pilots is that their cardiovascular systems adapt to frequent exposure to high +Gz levels. HYPOTHESIS: The purpose of this study was to demonstrate that the baroreflex-mediated cardiovascular response of a group of 8 fighter pilots to a mild accelerative stimulus differs from that of a group of 12 non-pilots. METHODS: Arterial pressures and heart rate responses to rapid head-up tilting to +75 degrees were compared between the two groups. Five variables were examined: systolic, diastolic, mean arterial and pulse pressures, and heart rate. RESULTS: The cardiovascular responses of the groups were fundamentally different. In response to tilt, the non-pilots showed little change in systolic and mean arterial pressures, diastolic pressure increased slightly and pulse pressure decreased significantly. In the pilot group, however, significant increases were observed in systolic, diastolic and mean arterial pressures (p < 0.05), whereas pulse pressure did not change. Between groups there were significant differences observed in terms of arterial pressures (p < 0.05), but not heart rate. CONCLUSION: The baroreflex of a fighter pilot is capable of adapting to +Gz, becoming a more sensitive and effective BP control system as a result. This adaptation affords the pilot greater protection against G-LOC.

Adult↗
PubMed1997

The structure of ribosomal protein S7 at 1.9 A resolution reveals a beta-hairpin motif that binds double-stranded nucleic acids.

BACKGROUND: Ribosomal protein S7, a crucial RNA-binding component of the ribosome, is one of two proteins that initiates assembly of the 30S ribosomal subunit. It is required for proper folding of a large 3' domain of 16S ribosomal RNA. S7 regulates its own synthesis by binding to its own mRNA. This ability of S7 to bind both messenger and ribosomal RNAs makes determination of its mode of RNA recognition particularly interesting. RESULTS: The crystal structure of S7 from Thermus thermophilus was determined by a two-wavelength anomalous diffraction experiment using the LIII edge of mercury. The S7 structure consists of a bundle of six helices and an extended beta hairpin between helices 3 and 4, with two or more RNA-binding sites on its surface. The hairpin, along with portions of helices 1, 4 and 6, forms a large, positively charged, concave surface that has the appropriate curvature and dimensions to bind double-stranded RNA. A second putative RNA-binding site comprises parts of loop 2 and the helix 4-loop 5 turn. CONCLUSIONS: Structural similarity between S7 and the IHF/HU family of proteins strongly suggests that the beta hairpin of S7 binds to a groove of double-stranded RNA. The beta hairpin of S7 is also similar to those from other nucleic acid binding proteins, such as ribosomal protein L14 and BIV Tat, suggesting that it belongs to an extended family of such motifs, all of which bind to a groove of double-stranded nucleic acid. The residues in S7 loop 2 that belong to the second putative RNA-binding site may have a role analogous to the N-terminal residues of IHF/HU which grip an unbent portion of double helix.

Amino Acid Sequence↗
PubMed1997

The activation domain of the MotA transcription factor from bacteriophage T4.

Bacteriophage T4 encodes a transcription factor, MotA, that binds to the -30 region of middle-mode promoters and activates transcription by host RNA polymerase. We have solved the structure of the MotA activation domain to 2.2 A by X-ray crystallography, and have also determined its secondary structure by NMR. An area on the surface of the protein has a distinctive patch that is populated with acidic and hydrophobic residues. Mutations within this patch cause a defective T4 growth phenotype, arguing that the patch is important for MotA function. One of the mutant MotA activation domains was purified and analyzed by NMR, and the spectra clearly show that the domain is properly folded. The mutant full-length protein appears to bind DNA normally but is deficient in transcriptional activation. We conclude that the acidic/hydrophobic surface patch is specifically involved in transcriptional activation, which is reminiscent of eukaryotic acidic activation domains.

Amino Acid Sequence↗
PubMed1996

Ribosomal protein L9: a structure determination by the combined use of X-ray crystallography and NMR spectroscopy.

The structure of protein L9 from the Bacillus stearothernophilus ribosome has been determined at 2.5 A resolution by refinement against single crystal X-ray diffraction data with additional constraints provided by NMR data. This highly elongated protein consists of two domains separated by a nine-turn connecting helix. Conserved aromatic and positively charged amino acid residues on the surface of each domain are likely to be directly involved in binding 23 S ribosomal RNA. The shape of the protein, with its two widely spaced RNA-binding sites, suggests that it may serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of the ribosomal RNA. The combined use of X-ray and NMR data in the refinement procedure was essential in defining the N-terminal domain of the protein, which was relatively poorly determined by the X-ray data alone. In addition to resolving the ambiguities in defining the hydrophobic core and side-chain conformations with the N-terminal domain, this combined NMR-X-ray analysis provides the first detailed and accurate view of the N-terminal RNA-binding site. NMR data also showed that the N-terminal domain is stable in solution, indicated by amide protons that are protected from solvent exchange. The lack of definition of the N-terminal domain in the X-ray structure is therefore likely due to packing disorder within the crystal rather than structural instability. This combined NMR-X-ray analysis provides a useful model as to how X-ray and NMR data can be practically and logically combined in the determination of the structure of a single protein molecule.

Amino Acid Sequence↗
PubMed1996

Helicase-contrahelicase interaction and the mechanism of termination of DNA replication.

Termination of DNA replication at a sequence-specific replication terminus is potentiated by the binding of the replication terminator protein (RTP) to the terminus sequence, causing polar arrest of the replicative helicase (contrahelicase activity). Two alternative models have been proposed to explain the mechanism of replication fork arrest. In the first model, the RTP-terminus DNA interaction simply imposes a polar barrier to helicase movement without involving any specific interaction between the helicase and the terminator proteins. The second model proposes that there is a specific interaction between the two proteins, and that the DNA-protein interaction both restricts the fork arrest to the replication terminus and determines the polarity of the process. The evidence presented in this paper strongly supports the second model.

Adenosine Triphosphatases↗
PubMed1996

Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

The replication terminator protein (RTP) of Bacillus subtilis is a homodimer that binds to each replication terminus and impedes replication fork movement in only one orientation with respect to the replication origin. The three-dimensional structure of the RTP-DNA complex needs to be determined to understand how structurally symmetrical dimers of RTP generate functional asymmetry. The functional unit of each replication terminus of Bacillus subtilis consists of four turns of DNA complexed with two interacting dimers of RTP. Although the crystal structure of the RTP apoprotein dimer has been determined at 2.6-A resolution, the functional unit of the terminus is probably too large and too flexible to lend itself to cocrystallization. We have therefore used an alternative strategy to delineate the three dimensional structure of the RTP-DNA complex by converting the protein into a site-directed chemical nuclease. From the pattern of base-specific cleavage of the terminus DNA by the chemical nuclease, we have mapped the amino acid to base contacts. Using these contacts as distance constraints, with the crystal structure of RTP, we have constructed a model of the DNA-protein complex. The biological implications of the model have been discussed.

Bacillus subtilis↗
PubMed1996

Structural evidence for specific S8-RNA and S8-protein interactions within the 30S ribosomal subunit: ribosomal protein S8 from Bacillus stearothermophilus at 1.9 A resolution.

BACKGROUND: Prokaryotic ribosomal protein S8 is an important RNA-binding protein that occupies a central position within the small ribosomal subunit. It interacts extensively with 16S rRNA and is crucial for the correct folding of the central domain of the rRNA. S8 also controls the synthesis of several ribosomal proteins by binding to mRNA. It binds specifically to very similar sites in the two RNA molecules. RESULTS: S8 is divided into two tightly associated domains and contains three regions that are proposed to interact with other ribosomal components: two potential RNA-binding sites, and a hydrophobic patch that may interact with a complementary hydrophobic region of S5. The N-terminal domain fold is found in several proteins including two that bind double-stranded DNA. CONCLUSIONS: These multiple RNA-binding sites are consistent with the role of S8 in organizing the central domain and agree with the latest models of the 16S RNA which show that the S8 location coincides with a region of complicated nucleic-acid structure. The presence in a wide variety of proteins of a region homologous to the N-terminal domain supports the idea that ribosomal proteins must represent some of the earliest protein molecules.

Amino Acid Sequence↗
PubMed1996

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif.

Amino Acid Sequence↗
PubMed1996

The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication.

The replication terminator protein (RTP) of Bacillus subtilis causes polar fork arrest at replication termini by sequence-specific interaction of two dimeric proteins with the terminus sequence. The crystal structure of the RTP protein has been solved, and the structure has already provide valuable clues regarding the structural basis of its function. However, it provides little information as to the surface of the protein involved in dimer-dimer interaction. Using site-directed mutagenesis, we have identified three sites on the protein that appear to mediate the dimer-dimer interaction. Crystallographic analysis of one of the mutant proteins (Y88F) showed that its structure is unaltered when compared to the wild-type protein. The locations of the three sites suggested a model for the dimer-dimer interaction that involves an association between two beta-ribbon motifs. This model is supported by a fourth mutation that was predicted to disrupt the interaction and was shown to do so. Biochemical analyses of these mutants provide compelling evidence that cooperative protein-protein interaction between two dimers of RTP is essential to impose polar blocks to the elongation of both DNA and RNA chains.

Bacillus subtilis↗
PubMed1996

Solution structure of prokaryotic ribosomal protein S17 by high-resolution NMR spectroscopy.

The solution of a primary 16S rRNA-binding ribosomal protein, S17, was investigated by two- and three-dimensional homonuclear and heteronuclear magnetic resonance spectroscopy. Almost complete chemical shift assignments for the 1H, 15N, and 13C resonances have been obtained. The NMR data have been rigorously analyzed using a combination of distance geometry, back-calculation, and simulated annealing refinement techniques, and a high-resolution three-dimensional structure has been deduced. The protein consists of a single twisted antiparallel beta-pleated sheet with Greek-key topology. The five beta-strands are connected by extended loops that are flexible compared to the beta-sheet core structure and appear not to adopt one definite conformation in solution. Two of these loops contain many of the residues that have been implicated in binding ribosomal RNA. The location and distribution of these residues and other positively charged side chains on the protein surface suggest an interaction with two distinct regions of ribosomal RNA.

Amino Acid Sequence↗
PubMed1996

The crystal structure of ribosomal protein L14 reveals an important organizational component of the translational apparatus.

BACKGROUND: Detailed structural information on ribosomal proteins has increased our understanding of the structure, function and evolution of the ribosome. L14 is one of the most conserved ribosomal proteins and appears to have a central role in the ribonucleoprotein complex. Studies have indicated that L14 occupies a central location between the peptidyl transferase and GTPase regions of the large ribosomal subunit. RESULTS: The crystal structure of L14 from Bacillus stearothermophilus has been solved using a combination of isomorphous replacement and multiwavelength anomalous dispersion (MAD) methods. The structure comprises a five-stranded beta-barrel, a C-terminal loop region that contains two small alpha-helices, and a beta-ribbon that projects from the beta-barrel. An analysis of the structure and the conserved amino acids reveals three surface patches that probably mediate L14-RNA and L14-protein interactions within the ribosome. CONCLUSIONS: The accepted role of ribosomal proteins is to promote the folding and stabilization of ribosomal RNA. The L14 structure is consistent with this notion, and it suggests that the RNA binds in two sites. One RNA-binding site appears to recognize a distinct region of ribosomal RNA during particle assembly. The second site is smaller and may become occupied during the later compaction of the RNA. The surface hydrophobic patch is a likely site of protein-protein interaction, possibly with L19.

Amino Acid Sequence↗
PubMed1995

NMR analysis of the trans-activation response (TAR) RNA element of equine infectious anemia virus.

Transcription of lentiviral DNA in the host cell is regulated by an interaction between the viral TAR RNA stem-loop and the viral Tat protein. Here we present a model of the three-dimensional structure of the TAR RNA stem-loop of the equine infectious anemia virus (EIAV), derived from two- and three-dimensional NMR data. This 25 nucleotide RNA consists of an A-form helical stem capped by two U-G base pairs and a four-nucleotide loop. Two loop cytidines are stacked into the loop interior and likely form a non-Watson-Crick C-C base-pair. The two nucleotides at the top of the loop, U13 and G14, appear to be excluded from the interior of the loop and solvent exposed. It is significant that now for the EIAV TAR-Tat system, three-dimensional structures are now known for both the RNA and protein components.

Base Sequence↗
PubMed1995

Crystal structure of the replication terminator protein from B. subtilis at 2.6 A.

The crystal structure of the replication terminator protein (RTP) of B. subtilis has been determined at 2.6 A resolution. As previously suggested by both biochemical and biophysical studies, the molecule exists as a symmetric dimer and is in the alpha + beta protein-folding class. The protein has several uncommon features, including an antiparallel coiled-coil, which serves as the dimerization domain, and both an alpha-helix and a beta-ribbon suitably positioned to interact with the major and minor grooves of B-DNA. A site has been identified on the surface of RTP that is biochemically and positionally suitable for interaction with the replication-specific helicase. Other features of the structure are consistent with the polar contrahelicase mechanism of the protein. A model of the interaction between RTP and its cognate DNA is presented.

Amino Acid Sequence↗