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At least 1,099 records · Page 61Linked to original sources
PubMed1997

Polyadenylated mRNA in Escherichia coli: modulation of poly(A) RNA levels by polynucleotide phosphorylase and ribonuclease II.

The effect of 3'-exoribonucleases on the polyadenylation of mRNA in Escherichia coli was studied by comparing the synthesis and levels of poly(A) RNA in wild-type E coli and mutant strains defective in the two major 3'-exoribonucleases: polynucleotide phosphorylase and ribonuclease II. Mutations which substantially reduced the activity of these 3'-exonucleases caused a 10-fold increase in pulse-labeling of total poly(A) RNA in intact cells. When the net rate of RNA synthesis was measured in permeabilized cells, the mutant with defective 3'-exonucleases showed 20- to 60-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes in non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable cells under conditions when 3'-exoribonucleases were inactive showed a 6-fold higher rate of poly(A) synthesis in the exonuclease-deficient mutant strain, suggesting a higher concentration of mRNA 3'-ends amenable to polyadenylation. Steady-state levels of poly(A) RNA, measured by the ability to serve as template for oligo(dT)-dependent complementary DNA synthesis, also increased more than 40-fold when the 3'-exonucleases were inactivated. Monitoring of the length of the poly(A) tracts by denaturing polyacrylamide gel electrophoresis showed chain lengths of up to 45 residues in the 3'-exonuclease-deficient mutant, whereas most of the poly(A) tracts in the parent strain were shorter than 12 residues. These results show that 3'-exonucleases reduce the level of polyadenylated mRNA in E coli not merely by causing its degradation but also by reducing its rate of synthesis, presumably by competing with poly(A) polymerase for the 3'-ends of mRNA.

Cell Membrane Permeability↗
PubMed2003

Changes in Escherichia coli transcriptome during acclimatization at low temperature.

Upon cold shock Escherichia coli transiently stops growing and adapts to the new temperature (acclimatization phase). The major physiological effects of cold temperature are a decrease in membrane fluidity and the stabilization of secondary structures of RNA and DNA, which may affect the efficiencies of translation, transcription, and replication. Specific proteins are transiently induced in the acclimatization phase. mRNA stabilization and increased translatability play a major role in this phenomenon. Polynucleotide phosphorylase (PNPase) is one of the cold-induced proteins and is essential for E. coli growth at low temperatures. We investigated the global changes in mRNA abundance during cold adaptation both in wild type E. coli MG1655 and in a PNPase-deficient mutant. We observed a twofold or greater variation in the relative mRNA abundance of 20 genes upon cold shock, notably the cold-inducible subset of csp genes and genes not previously associated with cold shock response, among these, the extracytoplasmic stress response regulators rpoE and rseA, and eight genes with unknown function. Interestingly, we found that PNPase both negatively and positively modulated the transcript abundance of some of these genes, thus suggesting a complex role of PNPase in controlling cold adaptation.

Adaptation, Physiological↗
PubMed2002

Running rings around RNA: a superfamily of phosphate-dependent RNases.

The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA. Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH. These enzymes are phosphorylases that degrade RNA from the 3'-end. A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily. Here, information from the structure of PNPase is used to address two related issues. First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme. Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction.

Amino Acid Sequence↗
PubMed1997

Spectroscopic study of the interaction of pazelliptine with nucleic acids.

The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2-6) x 10(5)M(-1). The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA, reveal a change in the protonation state of the drug upon binding, increasing the apparent pKa of its 9-N-nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs (approximately 30% increase of phi(F) whereas it is quenched in the vicinity of G-C base pairs (approximately 90% decrease of phi(F)). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-poly(dG-dC), suggesting a binding of PZE to G-C rich regions.

Antineoplastic Agents↗
PubMed1997

Distance geometry based comparative modelling.

A distance geometry based protein modelling algorithm is presented which relies on the projection of simple model chain coordinates into Euclidean spaces with gradually decreasing dimensionality. Fast embedding was achieved by performing separate distance matrix projections on subsets of the model points. Structural equivalences between the unknown target and related proteins with known structures were deduced either from a mixed sequence/structure multiple alignment or from the output of various fold recognition (threading) approaches. These equivalences were mapped onto the model as structure-specific conserved C alpha atom distances and secondary structure assignments. Additional nonspecific distance restraints derived from general stereochemical properties of folded protein chains were used to guide the modelling process. The method quickly constructed a large number of low-resolution models which could then serve as starting conformations for full-atom refinement. Structure predictions for some targets in the 'Asilomar Challenge' (CASP2) are presented to illustrate potential applications of the approach.

Algorithms↗
PubMed2000

Molecular interactions and metal binding in the theophylline-binding core of an RNA aptamer.

An RNA aptamer containing a 15-nt binding site shows high affinity and specificity for the bronchodilator theophylline. A variety of base modifications or 2' deoxyribose substitutions in binding-site residues were tested for theophyllinebinding affinity and the results were compared with the previously determined three-dimensional structure of the RNA-theophylline complex. The RNA-theophylline complex contains a U6-A28-U23 base triple, and disruption of this A28-U23 Hoogsteen-pair by a 7-deaza, 2'-deoxy A28 mutant reduces theophylline binding >45-fold at 25 degrees C. U24 is part of a U-turn in the core of the RNA, and disruption of this U-turn motif by a 2'-deoxy substitution of U24 also reduces theophylline binding by >90-fold. Several mutations outside the "conserved core" of the RNA aptamer showed reduced binding affinity, and these effects could be rationalized by comparison with the three-dimensional structure of the complex. Divalent ions are absolutely required for high-affinity theophylline binding. High-affinity binding was observed with 5 mM Mg2+, Mn2+, or Co2+ ions, whereas little or no significant binding was observed for other divalent or lanthanide ions. A metal-binding site in the core of the complex was revealed by paramagnetic Mn2+-induced broadening of specific RNA resonances in the NMR spectra. When caffeine is added to the aptamer in tenfold excess, the NMR spectra show no evidence for binding in the conserved core and instead the drug stacks on the terminal helix. The lack of interaction between caffeine and the theophylline-binding site emphasizes the extreme molecular discrimination of this RNA aptamer.

Base Sequence↗
PubMed1995

Role of Tyr-22 in the binding of Pf3 ssDNA binding protein to nucleic acids.

A Tyr-22-->Phe-22 (Y22F) mutant of the single-strand DNA binding protein (ssDBP) of the filamentous phage Pf3 was obtained by site-directed mutagenesis. An alignment of protein sequences indicates that Tyr-22 of the Pf3 ssDBP corresponds to Tyr-26 of the fd g5p, a tyrosine within the DNA-binding loop. The mutant Y22F Pf3 protein had a CD spectrum very similar to that of native, wild-type Pf3 ssDBP and could bind to both DNA and RNA polymers. In CD titrations of poly[r(A)], poly[r(C)], and Pf3 ssDNA with the Y22F mutant, the saturation endpoints remained the same as for titrations performed with wild-type Pf3 ssDBP, indicating that the mutant protein retained the same n = 2 mode of binding as the wild-type protein. However, a second stoichiometric mode of binding at a ratio of one protein monomer to about four nucleotides (n = 4) was observed for titrations of these nucleic acids with the Y22F mutant protein. Both proteins showed only an n = 2 mode of binding to poly[d(A)], poly[d(C)], and poly[d(T)] and only an n = 3 mode of binding to poly[r(U)]. Distinctly different CD spectral changes of the nucleic acid were observed in titrations of poly[d(A)] with the Y22F mutant and the wild-type protein. Therefore, the mutant and wild-type ssDBP interact differently with some nucleic acids, depending on the base and sugar composition, providing evidence that Tyr-22 is indeed in the DNA-binding loop and may be important in the sequence discrimination of the binding of the Pf3 ssDBP.

Amino Acid Sequence↗
PubMed1993

Synthesis and properties of adenosine oligonucleotide analogues containing methylene groups in place of phosphodiester 5'-oxygens.

The ADP analogue in which the 5'-oxygen has been replaced by a methylene group can be prepared by condensing 5'-deoxy-5'-phosphonomethyladenosine with inorganic phosphate. This analogue readily polymerizes onto the primer A-A in the presence of the enzyme polynucleotide phosphorylase and either Mg2+ or Mn2+. The initial products are of the form A-A(-cA)n-cA (where "-" and "-c" stand for the normal phosphodiester linkage and the linkage in which the 5'-oxygen is replaced with the methylene group, respectively). Treatment of these with alkali yields adenosine 2'(3')-phosphate and the series (A(-cA)n-cA containing only phosphonomethylene linkages. The decamer A(-cA)8-cA interacts with two molecules of U(-U)8-U to form a triple-standard structure that has a stability similar to that exhibited by the analogous complex formed from A(-A)8-A and U(-U)8-U. This property, along with the resistance of these oligomer analogues toward nucleases that cleave phosphodiester linkages between the phosphorus and the 5'-oxygen, should provide a strong rationale for application of phosphonomethylene linkages in schemes for therapeutic drug design that use the antisense strategy.

Adenosine Diphosphate↗
PubMed1993

Thermodynamics of single-stranded RNA and DNA interactions with oligolysines containing tryptophan. Effects of base composition.

We have examined the thermodynamics of binding of a series of oligolysines (net charge z = +2 to +10) containing one, two, or three tryptophans to several single-stranded (ss) homo-polynucleotides [poly(A), poly(C), poly(I), poly(dU), poly(dT)] and duplex (ds) DNA in order to investigate the effects of peptide charge, tryptophan content, and polynucleotide base and sugar type. Equilibrium association constants, Kobs, were measured as a function of monovalent salt concentration (KCH3CO2) and temperature by monitoring the quenching of the peptide tryptophan fluorescence upon interaction with the polynucleotides, from which the dependence of delta G(o)obs, delta H(o)obs, and delta S(o)obs on [KCH3CO2] was obtained. As observed previously with poly(U) [Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932], the dependence of delta G(o)obs on [K+] for peptide binding to each polynucleotide is entirely entropic in origin (i.e., delta H(o)obs is independent of [K+]), consistent with the conclusion that Kobs increases with decreasing salt concentration due to the favorable increase in entropy resulting from the displacement of bound cations (K+) from the nucleic acid upon formation of the complex. For each ss polynucleotide, we find that significantly less than one potassium ion is released thermodynamically per net positive peptide charge, as determined from the value of delta log Kobs/delta log [K+]. Interestingly, (-delta log Kobs/delta log [K+])/z decreases with increasing peptide charge for poly(A), poly(C), and poly(dT), contrary to the behavior observed with poly(U) and ds-DNA, which may reflect a significant release of bound water upon formation of peptide complexes with these ss homo-polynucleotides or an increased binding of K+ to the ss polynucleotide with increasing [K+]. Alternatively, there may be conformational differences between the bound states of oligolysines of low charge, relative to oligolysines of higher charge. However, in all cases, peptides with z < +4 display different thermodynamics of binding than peptides with z > +4. The presence of tryptophan (Trp) within these peptides does not influence the salt dependence of Kobs for binding to poly(A), poly(C), or poly(dT). However, the Trp content of the peptide does contribute significantly to the thermodynamics of these interactions: Trp interactions result in a favorable contribution to delta H(o)obs, but an unfavorable contribution to delta S(o)obs, with little effect on delta G(o)obs due to entropy-enthalpy compensations. Oligolysines containing Trp also display a small, but significant, dependence of Kobs on base composition, with Kobs decreasing in the order poly(I) >> poly(dT) approximately poly(U) approximately poly(A) >> poly(C).

Amino Acid Sequence↗
PubMed1994

Sequence specific thermodynamic and structural properties for DNA.RNA duplexes.

DNA.RNA hybrid duplexes are found in many important biological processes and are involved in developing modes of disease treatment, such as antisense therapy, yet little is known about the sequence dependence of their structure and stability. The structure and thermodynamic stability of DNA.RNA hybrid model systems corresponding in composition and length and containing (1) all purine or all pyrimidine bases on each strand or (2) mixed purine and pyrimidine bases on each strand have been evaluated relative to pure RNA and DNA duplexes by thermal melting, CD, and electrophoresis analyses. The spread in free energies of denaturation of the homopurine.homopyrimidine systems covers over 14 kcal/mol of single strands, while the mixed sequence free energies vary by less than 4 kcal/mol. The RNA-homopurine.DNA-homopyrimidine hybrid resembles a corresponding pure RNA duplex in both structure and stability, whereas the DNA-homopurine.RNA-homopyrimidine hybrid resembles a corresponding pure DNA duplex. The mixed sequence hybrids show intermediate structure between the corresponding pure RNA and pure DNA duplexes and a stability closer to that of the pure DNA duplex. From these results and the evaluation of published hybrid data [Hall, K. B., & McLaughlin, L. W. (1991) Biochemistry 30, 10606-10613; Roberts, W. R., & Crothers, D. M. (1992) Science 258, 1463-1466], it can be predicted that a hybrid duplex containing more RNA purine bases will have a CD spectrum, and probably conformation, resembling that of A-form duplexes and will be more stable than a corresponding hybrid duplex with fewer RNA purine bases.

Base Sequence↗
PubMed1993

Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics.

The duplex (rGGCGAGCC)2 contains tandem G x A mismatches--a common motif in the secondary structures of biological RNAs. The three-dimensional structure of (rGGCGAGCC)2 was derived using molecular dynamics and energy minimization with NMR-derived restraints for 78 interproton distances (per strand), 18 hydrogen bonds for the six Watson-Crick G x C pairs, and 26 dihedral angles (per strand). The G x A mismatch structures are similar to those observed in a DNA duplex [Li, Y., Zon, G., & Wilson, W. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 80, 26-30] and an RNA hairpin [Heus, H. A., & Pardi, A. (1991) Science 253, 191-193], with hydrogen bonds from guanine 2-amino and N3 to adenine N7 and 6-amino, respectively. The other G 2-amino and A 6-amino protons are within hydrogen-bonding distance of a phosphate oxygen and 2'-oxygen, respectively. Strong interstrand A-A and G-G stacking is observed between the G x A mismatches. This contrasts with the poor stacking observed between the G x A mismatches and closing G x C base pairs. The stems are basically A-form with all bases in the anti conformation and all nonterminal sugars in the C3'-endo conformation. The structure rationalizes previous thermodynamic, circular dichroism, and imino proton NMR results and suggests tandem G x A mismatches in RNA may provide a contact site for tertiary interactions.

Base Sequence↗
PubMed1991

Interactions of the A1 heterogeneous nuclear ribonucleoprotein and its proteolytic derivative, UP1, with RNA and DNA: evidence for multiple RNA binding domains and salt-dependent binding mode transitions.

The 319-residue A1 heterogeneous nuclear ribonucleoprotein is the best studied of the group of major or core mammalian hnRNP proteins that bind pre-mRNA immediately following transcription. Circular dichroism studies suggest that binding of A1 and its proteolytic fragment, UP1 (residues 1-195), to nucleic acids results in an unstacking of the bases of poly(A). On the basis of poly[d(A-T)] and poly[r(A-U)] melting studies, both A1 and UP1 are helix-destabilizing proteins. Titrations of A1 and UP1 with poly(A), poly(U), and poly[d(T)] suggest that these two proteins do not bind with significant base specificity. A previous study indicated that A1, which contains a glycine-rich COOH terminus (residues 196-319) not present in UP1, binds cooperatively to polynucleotides while UP1 does not [Cobianchi et al. (1988) J. Biol. Chem. 263, 1063-1071]. Here we confirm this latter finding and demonstrate that the cooperativity parameter for A1 binding, which has a value of about 35 for binding to both single-stranded RNA and DNA, is insensitive to the NaCl concentration at least up to 0.4 M. In contrast to the cooperativity parameter, the occluded site size for A1 binding to RNA is salt dependent and increases from about 14 to 28 upon increasing the NaCl concentration from 25 to 250 mM. This variation in site size is best explained by assuming that A1 can interact with nucleic acids via at least two different binding modes. Both A1 and UP1 have higher affinity for single-stranded as opposed to double-stranded nucleic acids and bind preferentially to single-stranded RNA as compared to DNA. Comparative studies on the binding of A1 versus UP1 to poly[r(epsilon A)] demonstrate that in addition to cooperative protein/protein interactions, the glycine-rich COOH-terminal domain of A1 is also directly involved in protein/nucleic acid interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

3'-end formation at the phage lambda tR1 rho-dependent transcription termination site.

The rho-dependent transcription terminator tR1 of bacteriophage lambda stops RNA synthesis downstream of the major rightward promoter, PR, shortly after the cro gene. Terminated transcripts produced in a purified in vitro transcription system display a heterodisperse set of 3' termini, occurring in clusters located at +290-300, 308-312, 340-345, 385-390, and 440-450 nucleotides from the transcription start site [Morgan, W.D., Bear, D.G., & von Hippel, P.H. (1983) J. Biol. Chem. 258, 9553-9564]. However, transcripts from the same promoter in vivo have been reported to end primarily at +310-312 [Court, D., Brady, C., Rosenberg, M., Wulff, D. L., Behr, M., Mahoney, M., & Izumi, S. (1980) J. Mol. Biol. 138, 231-254]. In order to understand the nature of this discrepancy, we have carried out a comparative analysis of lambda PR transcription products produced in translationally active S30 cell extracts, in a purified in vitro system and in vivo. RNAs from the cell extracts coupled to translation show primarily three PR-derived transcripts beginning at one predominant 5' end and terminating at +263, 308, and 318. Sites +263 and +308 appear to be RNA processing sites. S1 nuclease mapping studies of RNAs produced in vivo show one 5' end and two 3' termini ending at +263 and 311; the +263 site is the predominant 3' end. When transcripts produced in a purified in vitro transcription system are incubated in the S30 cell extract under various conditions, the RNAs are degraded to two primary products with lengths of 263 and 308-311 nt.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1982

Comparative studies on ribonucleic acid dependent RNA polymerases in cucumber mosaic virus infected cucumber and tobacco and uninfected tobacco plants.

RNA-dependent RNA polymerases have been isolated in almost pure form from cucumber mosaic virus (CMV) infected cucumber cotyledons and from tobacco leaves and were compared with the less pure enzyme from uninfected tobacco. The purified polymerase from cucumber shows on sodium dodecyl sulfate gels two peptide chains of about 100 and 112 kdaltons. The enzyme from tobacco shows a close doublet of about 125 kdaltons, which is also present in the less pure preparation from healthy tobacco. While both the cucumber and tobacco enzymes can use many polynucleotides and RNAs as templates, considerable quantitative differences exist, poly(C) being by far the most effective template for the cucumber enzyme but of low activity with the tobacco enzyme and poly(UG) being highly active with the latter but not the former. Poly(A) and poly(G) are inactive. Different viral RNAs, including CMV RNA, show smaller differences. The sedimentation rates of the enzyme from both sources are the same as that of gamma-globulin. A uridine 5'-triphosphate (UTP) terminal transferase also present in both plants sediments much more slowly and can be completely removed from the RNA polymerases. However, slight nucleolytic activity remains associated with the purified polymerases and appears to be proportional to the polymerase activity. The conclusion derived from these data is that the RNA-dependent RNA polymerases of different plants differ and are not detectably affected by virus infection in qualitative terms while being produced in greatly increased amounts upon some virus infections. Similar conclusions were previously reached with less purified enzyme preparations from tobacco as compared to cowpea, infected with different viruses, if any.

Kinetics↗
PubMed1982

Function of transcription termination factor rho in a model transcription system using synthetic deoxyribonucleic acid as template.

The function of a transcription termination factor, rho, has been studied by using several synthetic DNAs with simple repetitive base sequences as templates for transcription. rho actually exhibits various effects on transcription depending on the base sequence of the template: (1) rho terminates poly(A) synthesis with poly(dA) x poly(dT), poly(dT), or oligo(dT), leading to release of RNA from RNA polymerase. rho also inhibits the synthesis of other homoribopolymers such as poly(U) directed by poly(dA) x poly(dT) and poly(C) and poly(I) directed by poly(dG) x poly(dC), presumably by a similar mechanism. (2) rho inhibits the synthesis of another homoribopolymer, poly(G), directed by poly(dG) x poly(dC) at the step of initiation rather than propagation of transcription. (3) rho stimulates rather than inhibits the synthesis of poly(A-C) and poly(G-U) directed by poly[d(A-C)] x poly[d(G-T)], presumably by enhancing the dissociation of transcription complexes. (4) rho has no influence on the synthesis of poly(A-U) and poly(G-C) directed by poly[d(A-T)] and poly[d(G-C)], respectively. In the first case, but not otherwise, the effect of rho is coupled with its RNA-dependent nucleosidetriphosphate phosphohydrolase activity, as is rho-mediated transcription termination on natural templates. The implication of these results is discussed in reference to the current view that rho acts on transcription complexes that have ceased elongation and causes release of RNA in an energy-requiring reaction.

Base Sequence↗