Biomedical subjects

E G Wagner

Publications and source records attributed to E G Wagner.

46 records · Page 3Linked to original sources

PubMed1990

Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT.

The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation. Synthesis of RepA is negatively regulated both at the transcriptional and post-transcriptional levels. Post-transcriptional control is exerted through the action of an antisense RNA, CopA RNA. The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA. Binding between CopA and CopT inhibits repA expression. We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs. In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps. The first step is the formation of an initial, transient complex; stem-loop II is the structure in CopA necessary and sufficient for this step. The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single-stranded nucleotides located 5' to stem-loop II in CopA, and its counterpart in CopT. We show that the single-stranded region can be positioned on either side of stem-loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence-specific. Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra-strand breaking and inter-strand formation of hydrogen bonds.

Bacterial Proteins↗

PubMed1990

Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing.

The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA. Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT. We have previously shown that the in vitro formation of the CopA-CopT duplex follows second-order kinetics and occurs in at least two steps. The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex. Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex. Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k-1) for the kissing complex (which are inferred to be the same for CopI-T and CopA-T), and measured the hybridization rate constant k2 (for CopA-T k2 is at least 1000-fold greater than for CopI-T). The analysis of CopA derivatives of mutant and wild-type origin shows that the rate of formation of the kissing complex is rate-limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo. The biological implications of the kinetically irreversible RNA-RNA binding reaction scheme are discussed.

DNA Mutational Analysis↗

PubMed1989

Secondary structure analysis of the RepA mRNA leader transcript involved in control of replication of plasmid R1.

The main replication control function in plasmid R1 is an antisense RNA, CopA RNA. By binding to its target (CopT) in the leader of the RepA mRNA, CopA RNA inhibits the expression of the rate-limiting RepA protein. The formation of the RNA duplex has been proposed to alter the folding around the RepA start region. Knowledge of the secondary structure of both CopA and CopT RNA is crucial for an understanding of the regulation. Previously, we reported the structure of CopA RNA under native conditions. In the present communication we have analyzed the secondary structure of the RepA leader transcript. Our main findings are: The two loops of CopA RNA have their correspondence in CopT RNA. No major structural changes are found downstream of the duplex when CopA was bound to its target RNA during transcription. Furthermore, in agreement with CopA/CopT RNA binding studies reported recently we do not find evidence for the existence of a binding window.

Bacterial Proteins↗

PubMed1988

Translational control by antisense RNA in control of plasmid replication.

Control of replication of plasmids involves two processes: measurement of the copy number of the plasmid and adjustment of the replication frequency accordingly. For both these processes IncFII plasmids use an antisense RNA (CopA RNA) that forms a duplex with the upstream region (CopT) of the mRNA of the rate-limiting RepA protein. The kinetics of duplex formation was measured in vitro for the wild type and for a cop mutant plasmid; the mutant showed a reduction in the second-order rate constant for the formation of the RNA duplex and a similar increase in copy number. Hence, the kinetics of duplex formation and the concentration of CopA RNA determines the copy number of the plasmid.

Base Sequence↗

PubMed1988

Control of replication of plasmid R1: kinetics of in vitro interaction between the antisense RNA, CopA, and its target, CopT.

The frequency of replication of IncFII plasmids is regulated by the availability of a rate-limiting protein, RepA. This protein acts to promote initiation of replication and its synthesis is negatively controlled both at the transcriptional and translational level. The translational control is exerted by the binding of a small antisense RNA, CopA RNA, to its target, CopT, which is located in the leader region of the RepA mRNA. As a consequence, formation of RepA is inhibited. Here we demonstrate the binding of CopA RNA to CopT RNA in vitro; the rate constant of binding was determined to be approximately 1 X 10(6) M-1 s-1 at 37 degrees C. We have also shown that in vitro synthesized RepA mRNA molecules differing in length, but which contain the whole CopT region, are able to bind CopA RNA with similar rates. Analysis of the binding of CopA/CopT molecules derived from a copy-number mutant plasmid showed that the effect of the mutation on the rate of in vitro binding correlates well with its phenotypic effect in vivo, i.e. the binding rate constant is lowered in proportion to the increase in copy number. Likewise, the result of an in vitro incompatibility test is in agreement with in vivo data.

DNA Replication↗

PubMed1987

Control of replication of plasmid R1: translation of the 7k reading frame in the RepA mRNA leader region counteracts the interaction between CopA RNA and CopT RNA.

Replication of IncFII plasmids is regulated through the expression of a gene, repA. The RepA protein is rate-limiting for initiation of replication. The main negative control is exerted by a countertranscript, CopA RNA, that binds to the complementary region of the RepA mRNA, thereby inhibiting the formation of the RepA protein. The target region for CopA RNA, CopT, is located upstream of the RepA coding region. An open reading frame for a putative 7k protein overlaps the CopT sequence. Here we show by using lacZ fusions that the 7k gene is expressed. We constructed a translation start mutation in order to abolish formation of the 7k protein. This resulted in a 10-fold decrease in repA expression. The 7k protein produced in trans did not reverse this effect, so the 7k protein per se does not control expression of repA. However, translation of the 7k coding sequence must influence CopA/CopT RNA recognition, since ribosomes will transiently disrupt the target hairpin. We propose here a novel mechanism that affects the level of gene expression: the 7k region of the RepA mRNA is a leader sequence that is involved in expression of the downstream gene; translation of the 7k region competes with a negative control system involving RNA-RNA interaction.

Amino Acid Sequence↗

PubMed1982

Kinetic suppression of translational errors by (p)ppGpp.

The effect of (p)ppGpp on the accuracy of translation was studied in vitro using a poly(U)-programmed poly(Phe)-synthesizing system operating at incorporation rates and missense error frequencies close to the values obtained in vivo. Simulation of a relaxed phenotype in vitro was accomplished by limitation of the cognate aminoacyl-tRNA synthetase, while the noncognate aminoacyl-tRNA synthetase was included at saturating concentrations. This protocol yielded a Phe-tRNA-starved steady state system displaying the expected decrease in Phe polymerization rates accompanied by a drastic increase in relative Leu misincorporation errors. The use of purified enzymes permitted us to assay for the effects of the individual nucleotides ppGpp and ppGpp as well as their potential targets, the elongation factors Tu and G, upon the missense error rates. Our results support the conclusion that ppGpp reduces misincorporation in a starved in vitro system by preferentially inhibiting EF-Tu. The details of the proposed mechanism and their relevance to an in vivo situation are discussed.

Cell-Free System↗

PubMed1982

Rate of elongation of polyphenylalanine in vitro.

Ribosomes purified from Escherichia coli were preincubated with AcPhe-tRNA and poly(U). Then purified components necessary for polypeptide synthesis were added. Incubation of the complete system led to a burst of elongation which lasted for nearly 10 s. During the initial burst approximately 10% of the ribosomes participated in the elongation of poly(Phe) at an average rate per ribosome close to eight peptide bonds/s. The missense error rate with leucine was 4 x 10(-4) during the burst. Accordingly, the preincubated elongation system functions at a rate, as well as an accuracy, close to those of protein synthesis in vivo.

Amino Acyl-tRNA Synthetases↗

PubMed1980

Escherichia coli elongation factor G blocks stringent factor.

The relationship between the binding domains of elongation factor G(EF-G) and stringent factor (SF) on ribosomes was studied. The binding of highly purified, radioactively labeled, protein factors to ribosomes was monitored with a column system. The data show that binding of EF-G to ribosomes in the presence of fusidic acid and GDP or of the noncleavable analogue GDPCP prevents subsequent binding of SF to ribosomes. In addition, stabilization of the EF-G-ribosome complex by fusidic acid inhibits SF's enzymatic activities. Removal of protein L7/L12 from ribosomes leads to weaker binding of EF-G, while SF's binding and activity are unaffected. In the absence of L7/L12, EF-G-dependent inhibition of SF binding and function is reduced. The data presented in this report suggest that these two factors bind at overlapping, or at least interacting, ribosomal domains.

Escherichia coli↗

PubMed1980

Translational accuracy enhanced in vitro by (p)ppGpp.

The effect of (p)ppGpp on the accuracy of translation in vitro was studied with a system that has a missense error frequency similar to that of living bacteria. When poly (U) is translated, limitation of the system in Phe increases the Leu missense error frequency. The introduction of (p)ppGpp to the Phe-limited mixtures reduces significantly the missense errors as well as reduces the rate of translation. The introduction of (p)ppGpp to a full system has no effect on the accuracy of translation but does reduce its rate. The effects of (p)ppGpp on rate and accuracy of translation can be simulated in part by other inhibitors of translation such as GDPCP, fusidic acid and tetracycline. Furthermore, the presence of ppGpp or GDPCP in a Phe-limited system leads to an accumulation of Phe-tRNA, while a Phe-limited system that contains only GTP has negligibly small concentrations of Phe-tRNA. We conclude that one way in which (p)ppGpp improves the accuracy of translation is by permitting the system to maintain a favorable Phe-tRNA/Leu-tRNA ratio.

Cell-Free System↗