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At least 937 records · Page 52Linked to original sources
PubMed2006

Replication-associated gene dosage effects shape the genomes of fast-growing bacteria but only for transcription and translation genes.

The bidirectional replication of bacterial genomes leads to transient gene dosage effects. Here, we show that such effects shape the chromosome organisation of fast-growing bacteria and that they correlate strongly with maximal growth rate. Surprisingly the predicted maximal number of replication rounds shows little if any phylogenetic inertia, suggesting that it is a very labile trait. Yet, a combination of theoretical and statistical analyses predicts that dozens of replication forks may be simultaneously present in the cells of certain species. This suggests a strikingly efficient management of the replication apparatus, of replication fork arrests and of chromosome segregation in such cells. Gene dosage effects strongly constrain the position of genes involved in translation and transcription, but not other highly expressed genes. The relative proximity of the former genes to the origin of replication follows the regulatory dependencies observed under exponential growth, as the bias is stronger for RNA polymerase, then rDNA, then ribosomal proteins and tDNA. Within tDNAs we find that only the positions of the previously proposed 'ubiquitous' tRNA, which translate the most frequent codons in highly expressed genes, show strong signs of selection for gene dosage effects. Finally, we provide evidence for selection acting upon genome organisation to take advantage of gene dosage effects by identifying a positive correlation between genome stability and the number of simultaneous replication rounds. We also show that gene dosage effects can explain the over-representation of highly expressed genes in the largest replichore of genomes containing more than one chromosome. Together, these results demonstrate that replication-associated gene dosage is an important determinant of chromosome organisation and dynamics, especially among fast-growing bacteria.

Bacteria↗
PubMed2001

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.

Animals↗
PubMed1994

Methionyl-tRNA synthetase needs an intact and mobile 332KMSKS336 motif in catalysis of methionyl adenylate formation.

The family of aminoacyl-tRNA synthetases may be split into two classes according to the occurrence of specific combinations of peptide motifs. This study deals with the functional role of the KMSKS motif, which, in association with the HIGH motif, defines class 1 aminoacyl-tRNA synthetases. Each residue in the 332KMSKS336 sequence of Escherichia coli methionyl-tRNA synthetase, as well as R337 and the two surrounding G330 and G338 residues, were mutagenized. The comparison of the kinetic and equilibrium parameters of the methionine activation reaction sustained by the resulting variants enables the following conclusions to be drawn. (1) Mutation of all the residues studied strongly destabilizes the transition state for the formation of methionyl adenylate whilst changing moderately the stability of the ground state ternary complex enzyme, methionine: ATP-Mg2+. The consequences of the mutations are also reflected at the level of the stability of the ground state enzyme, methionyl adenylate:PPi-Mg2+ complex which is systematically decreased. (2) The substitution with alanine of any one of the three basic residues K332, K335 and R337 destabilizes the transition state by more than 3.2 kcal/mol, while substitution of the non-basic residues M333, S334 or S336 destabilizes it by at most 2.5 kcal/mol. Such a difference may reflect different modes of action of the residues, with the basic ones directly interacting with the beta and gamma phosphates of the ATP-Mg2+ substrate and the non-basic ones playing a structural role and/or participating in mobility of the enzyme region carrying the motif. (3) Modification of G330 or G338 to a proline markedly decreases the kinetic rate of methionyl adenylate formation. This behaviour suggests that the flexibility of the KMSKS loop in the structure of methionyl-tRNA synthetase is required to reach the transition state during formation of methionyl adenylate.

Adenosine↗
PubMed2005

Optimizing splinted ligation of highly structured small RNAs.

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73 mer tRNA, the other a 49 mer synthetic ribozyme. A stable LCC could be isolated and turned over with>95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

Base Sequence↗
PubMed2001

Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the trp leader transcript. trpE translational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from trpE'-'lacZ translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of trpE translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for trpE and trpD overlap by 29 nucleotides, we examined expression levels from trpED'-'lacZ translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that trpE and trpD are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates trpD translation via translational coupling. We also constructed a rho mutation to investigate the role of Rho-dependent termination in trp operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.

Bacillus subtilis↗
PubMed2002

High-resolution structures of large ribosomal subunits from mesophilic eubacteria and halophilic archaea at various functional States.

Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins. Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains. Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA. However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame. Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles. The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain.

Anti-Bacterial Agents↗
PubMed1975

Physical and coding properties of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides.

This report concerns the synthesis of poly(5-aminouridylic acid) and of 5-aminouridine-containing trinucleotides. Starting from 5-aminouridine the nucleoside 5'-phosphate was prepared enzymatically with carrot phosphotransferase whereas the nucleoside 5'-diphosphate was prepared chemically and polymerised with polynucleotide phosphorylase. The aminouridine-containing trinucleotides were prepared by known enzymatic procedures. Besides an increase of stability in the secondary structure poly(nh25U) forms a triple-stranded complex with poly(A) and stimulates the poly(Phe) synthesis like poly(U). In contrast to U-nh25U-U, the triplet containing the 3'-terminal aminouridine does not stimulate the binding of Phe-tRNA to 70-S ribosomes. This behavior is discussed with respect to the influence of a modification on the stacking geometry of a codon and the base pairing scheme between the 5'-nucleotide of the anticodon and the 3'-nucleotide of the condon.

Escherichia coli↗
PubMed2004

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Animals↗
PubMed2005

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme.

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 A. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ0. This crystal form diffracted to 2.25 A resolution.

Bacillus subtilis↗
PubMed2000

In vitro transcription by the turnip yellow mosaic virus RNA polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases.

Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3' ACCA end. By mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest DeltaG value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.

Alfalfa mosaic virus↗
PubMed1980

Chinese hamster ovary cells resistant to beta-aspartylhydroxamate contain increased levels of asparagine synthetase.

The growth of Chinese hamster ovary cells in a complete medium lacking asparagine is inhibited by beta-aspartylhydroxamate. The inhibition is overcome by the presence of asparagine in the growth medium. beta-Aspartylhydroxamate inhibits the activity of both asparagine synthetase and asparaginyl-tRNA synthetase in vitro. beta-Aspartylhydroxamate-resistant clones of Chinese hamster ovary cells have been isolated and three of these have been characterized. One clone, AH12, is 3-fold more resistant to beta-aspartylhydroxamate than the parental line and has 2 times higher levels of asparagine synthetase activity. Strains AH2 and AH5 are 6- to 7-fold more resistant to beta-aspartylhydroxamate and have 5 times higher levels of asparagine synthetase. The regulation of the expression of asparagine synthetase is altered in all three resistant cell lines. Whereas asparagine synthetase activity varies 2- to 3-fold in response to the asparagine content of the medium or to the extent of aminoacylation of tRNALeu in the parental cells, the activity of asparagine synthetase in the resistant cells is elevated under all growth conditions. No significant changes in the Km for substrates, Ki for beta-aspartylhydroxamate, or thermal stability were found for the asparagine synthetase of the resistant cells. These variants should prove useful in understanding the mechanisms involved in regulating the levels of asparagine synthetase in mammalian cells.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Deletions in the large (beta) subunit of a hetero-oligomeric aminoacyl-tRNA synthetase.

Glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases in Escherichia coli that is comprised of heterologous subunits which are organized in an alpha 2 beta 2 quaternary structure. The two subunits are encoded by a single mRNA with the region for alpha (303 codons) subunit followed by that for beta (689 codons) subunit. Five COOH-terminal deletions in the beta subunit coding region have been created. Each deletion protein has been investigated for its synthesis and stability in vivo, adenylate synthesis activity in vitro, and aminoacylation activity in vivo and in vitro. This has been done in the presence of free alpha subunit and, additionally, with alpha subunit that is fused by its carboxyl terminus to the amino terminus of each of the beta subunit deletion proteins. With a fused or unfused alpha chain, over 100 amino acids can be deleted from the carboxyl terminus of the beta chain without loss of in vivo complementation of a delta glyS (deletion) strain. Further analysis shows that the alpha subunit and approximately the amino-terminal half of the beta subunit are sufficient for the adenylate synthesis activity. In particular, a deletion of 306 amino acids from the COOH terminus of the beta subunit has little effect on the Km parameter for ATP or glycine in the pyrophosphate exchange reaction. The tRNA-dependent step in aminoacylation requires additional beta subunit sequences on the COOH-terminal side of those needed for adenylate synthesis. In these respects, the functional organization of the beta chain parallels that of several aminoacyl-tRNA synthetases which have only homologous subunits. In the case of the glycine enzyme, however, the heterologous alpha subunit is required for the elucidation of activities encoded by functional determinants of the beta chain.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Ligand binding and enzymic catalysis coupled through subunits in tyrosyl-tRNA synthetase.

The interaction of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus with its substrates in the aminoacyl adenylation reaction has been studied by stopped-flow fluorescence. The observed changes have been assigned to their chemical and physical processes by comparison with equilibrium dialysis, pyrophosphate exchange kinetics and rapid quenching and sampling techniques to give the rate constants for ligand binding, the formation of tyrosyl adenylate, and the reverse reaction. The stoichiometry of tyrosine and ATP binding in the catalytic process has been determined directly by equilibrium dialysis and equilibrium gel filtration under pyrophosphate exchange conditions, i.e., where a steady state has been set up in which the equilibrium position favors starting materials. It is shown that the rate-determining step in the formation of tyrosyl adenylate involves 1 mole each of tyrosine and ATP. A second mole of tyrosine and ATP bind to the aminoacyl adenylate complex stabilizing the high-energy intermediate. The enzyme tyrosyl adenylate complex that is isolated by gel filtration is in a different conformational state from that in the presence of tyrosine and ATP.

Adenosine Monophosphate↗
PubMed1988

Functional dissection of 5' and 3' extragenic control regions of human tRNA(Val) genes reveals two different regulatory effects.

Two natural human tRNA(Val) genes, pHtV1 and pHtV3, differ in their transcription efficiency by an order of magnitude. The extragenic control regions (ECRs) responsible for this effect were compared with respect to the kinetics and thermodynamics of transcription complex formation. The 5' ECR of pHtV1 acts by increasing both the rate of stable complex formation and the equilibrium constant of association between tDNA and at least one transcription factor present in the stable complex. The stability of the preinitiation complexes is not affected by ECRs. For the formation of a stable preinitiation complex, we suggest a two-step mechanism, comprising (i) the ECR-controlled association of at least one transcription factor (TFIIIC) with the tDNA, and (ii) an ECR-independent conformational change of this tDNA-protein complex. The function of 3' ECRs could be discriminated from the 5' ECR-mediated effects by transcriptional analysis of two chimeric constructs derived from pHtV1 and pHtV3. Surprisingly, the pHtV1 3' ECR causes an eight-fold increase of transcription efficiency, although it has only minor influence on stable preinitiation complex formation. Instead, this ECR stimulates transcription by promoting the transition of the preinitiation complex into an activity synthesizing transcription complex. This novel function of a 3' ECR contributes an additional regulatory level for tRNA gene expression.

Base Sequence↗
PubMed1979

Real-time solvent exchange studies of the imino and amino protons of yeast phenylalanine transfer RNA by Fourier transform NMR.

Real-time solvent exchange measurements using Fourier transform NMR at 270 MHz are presented. By means of the fast gel filtration column techniques originally developed for tritium exchange experiments, we were able to replace the solvent of a tRNA sample from an 1H2O to an 2H2O buffer and obtain a useful spectrum in 2-5 min. At 15 degrees C, there are 5 +/- 1 lowfield (-11 to -15 ppm relative to 2,2-dimethyl-2-silapentane-5-sulfonate) imino protons with exchange half times of minutes to hours. In addition, the m7G-46 C(8) proton and several amino protons are observed to exchange with similar rates. Analogous studies on unfractionated yeast tRNA suggest that such a class of slowly exchanging imino protons is present in several tRNAs, and that the activation energy for exchange is small [[approximatley 5 kcal/mol (21 kJ/mol)]. We speculate that these imino resonances arise from D-stem protons and that their slow exchange reflects stabilization by the numerous tertiary interactions involving this stem and the Mg2+ bound at the P-10 bend.

Base Composition↗
PubMed2000

Limited proteolysis of yeast elongation factor 3. Sequence and location of the subdomains.

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.

Amino Acid Sequence↗
PubMed2006

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I.

Effects of tRNA(Ala)(UGC) and its derivative devoid of the 3'-ACCA motif [tRNA(Ala)(UGC)DeltaACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysed in vivo and in vitro. In an amino-acid-starved relA mutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenic relA(+) host. Overexpression of tRNA(Ala)(UGC), which under such conditions occurs in Escherichia coli mostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3'-ACCA-truncated tRNA(Ala)(UGC). Moreover, tRNA(Ala)(UGC), but not tRNA(Ala)(UGC)DeltaACCA, caused RNA I cleavage in vitro in the presence of MgCl(2). These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearing E. coli, and demonstrate that tRNA(Ala)(UGC) participates in specific degradation of RNA I in vivo and in vitro. This reaction is dependent on the presence of the 3'-ACCA motif of tRNA(Ala)(UGC).

Bacteriocin Plasmids↗