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R S Hayward

Publications and source records attributed to R S Hayward.

At least 55 records · Page 3Linked to original sources
PubMed1984

Evidence for rifampicin-promoted readthrough of a fully rho-dependent transcriptional terminator.

We have previously presented evidence that rifampicin promotes readthrough of various transcriptional terminators in vivo. This conclusion was based on measurements of galactokinase or beta-galactosidase synthesis in Escherichia coli strains, harbouring plasmids having the terminators fused upstream of galK or lacZ respectively. The terminators tested did not include any known example of a fully rho-dependent signal. We now show that the drug does stimulate galactokinase production when galK is fused downstream of such a terminator: the first rightward terminator of lambda. We also show that the nusA1 host mutation affects neither termination nor the drug response at this site.

Escherichia coli↗
PubMed1982

Evidence that rifampicin can stimulate readthrough of transcriptional terminators in Escherichia coli, including the attenuator of the rpoBC operon.

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli, rpoB and rpoC, lie downstream of at least two ribosomal protein genes, rplJ (encoding L10) and rplL (L7/12), in a common operon. All four genes are served by promoter PL10, and an attenuator (partial terminator) of transcription, t1, lies between rplJL and rpoBC. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can stimulate transcription of rpoBC. We have investigated the locus of this effect by fusing PL10 and t1 separately to galK, in suitable plasmids. Our studies of these fusions, and similar fusions involving transcriptional terminators derived from coliphage T7, indicate that low concentrations of rifampicin cause increased readthrough of several different transcriptional terminators in E.coli in vivo, including rpo t1. We discuss whether or not this unspecific mechanism is solely responsible for the observed stimulatory effects of the drug on rpoBC transcription.

Base Sequence↗
PubMed1982

Effect of rifampicin on expression of lacZ fused to promoters or terminators of the E.coli rpoBC operon.

The genes encoding the beta and beta' subunits of RNA polymerase in E.coli lie downstream of at least two ribosomal protein genes in a single unit of transcription. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can strongly stimulate transcription of the polymerase genes, while activating the neighbouring ribosomal genes only slightly. We have investigated the mechanism of this effect by fusing strong promoters, a weak internal promoter, and an attenuator of the polymerase operon to the lacZ gene, in derivatives of plasmid pMC81. Studies of these fusions confirm our conclusion, based on similar fusions to galK, that rifampicin can foster readthrough of transcriptional terminators. They also suggest the existence of extra terminators and anti-termination elements in the above transcription unit.

DNA Restriction Enzymes↗
PubMed1981

Termination of transcription of the coliphage T7 "early" operon in vitro: slowness of enzyme release, and lack of any role for sigma.

The leftmost portion of the coliphage T7 genome is transcribed by the RNA polymerase Escherichia coli immediately after infection. This "early" operon is delineated by three promoters on the left, and a transcriptional terminator on the right. The terminator is efficient both in vivo, and with highly purified RNA polymerase in vitro. We have studied termination in vitro, using synchronously initiated transcription reactions with low enzyme:DNA ratios. We show that recognition of the stop signal and release of RNA product are relatively rapid. Dissociation of the enzyme from the DNA is quite slow, and probably rate-limiting for re-cycling of the polymerase. It is well established that the sigma subunit of RNA polymerase is required for specific initiation, but redundant during RNA elongation. By exploiting antisigma antiserum we have obtained evidence that sigma is also redundant during all steps of termination in vitro.

Aminoglycosides↗
PubMed1981

A possible role in transcription for the single-stranded DNA binding protein of bacteriophage T7.

A spontaneous rifampicin-resistant mutant of E. coli, RpoB26, which inhibits the growth of bacteriophage T7, has been described in the accompanying paper (Schwarz et al.). The rifr mutation appears to increase the rate of transcriptional termination in a rho-deficient strain. T7 mutants with the ability to grow (Gor+) on the Rifr mutant were isolated, and some of their properties were investigated. One of these Gor+ mutants has a small deletion, located between nucleotides 9694 and 9820 of the T7 DNA sequence (Dunn and Studier 1980), which affects the size of the T7 single-stranded DNA binding protein (ssDBP), the product of gene 2.5 (p 2.5) (Dunn and Studier 1980). The Gor+ phenotype was also mapped to this region by genetic methods. Gor+ is recessive to the wild-type (Gor-) phenotype. This suggests that the T7 ssDBP may normally increase the frequency of transcriptional termination in the early region by binding to single-stranded nucleic acid configurations, and so affecting molecular conformations involved in the termination process. Excessive termination in RpoB26 could therefore be compensated for by alterations of the ssDBP.

Carrier Proteins↗
PubMed1981

Internal promoters of the rpoBC operon of Escherichia coli.

Four ribosomal protein genes, rplA, rplJ, rplK and rplL form an operon in E. coli together with the genes rpoB and rpoC which encode the beta and beta' subunits of RNA polymerase. Transcription is initiated principally at two promoters, PL11 and Pl, the overall structure of the operon being (in the direction of transcription) PL11 rplK rplA Pl rplJ rplL rpoB rpoC. Here we describe studies of phage lambda derivatives carrying various segments of this operon, which demonstrate the existence of at least three additional weak internal promoters, and help to define their positions and strengths.

Bacteriophage lambda↗
PubMed1980

Cloning of DNA of the rpoBC operon from the chromosome of Escherichia coli K12.

We provide evidence that, in terms of transcriptional organisation, the rpoBC operon carried by lambdarifd 18 accurately represents the corresponding region of the E. coli K12 chromosome. A restriction fragment of E. coli K12 chromosomal DNA carrying the genes rpoBC (encoding the beta and beta' subunits of RNA polymerase) and rplL (coding for ribosomal proteins L7/L12) was cloned in a lambda vector, and the resulting phage tested for gene expression. In common with the corresponding fragment of lambdarifd 18 DNA, the chromosomal fragment has no strong promoter for rplL or rpoBC transcription. Another new phage was constructed by adding, to the restriction fragment carrying the rplL rpoBC structural genes from lambdarifd 18, a sequence from the E. coli K12 chromosome which includes a promoter for these genes. As in lambdarifd 18 itself, this promoter is shared with rplJ but not with rplKA. The properties of the latter phage also show that the dominant rifampicin-resistance characteristic of lambdarifd 18 results from more than one mutation.

Bacteriophage lambda↗
PubMed1979

New genes and promoters suggested by the DNA sequence near the end of the coliphage T7 early operon.

We have employed the dideoxynucleotide chain-terminating method to determine the nucleotide sequence of T7 DNA between the physical map positions 18.9% and 19.8%. The most striking features of this sequence are two perfect 21-basepair repeats, each of which appears to contain a promoter for late transcription. In each case the promoter sequence incorporates a putative translational terminator on its left (5'-side of the "sense" strand), and overlaps a potential ribosome-binding site on its right. The region probably lies immediately distal to the early operon, and may contain two short, hitherto unreported protein-coding sequences.

Base Sequence↗
PubMed1979

Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli.

The adjacent genes rpoB and rpoC code for the beta and beta' subunits of RNA polymerase in Escherichia coli, and are cotranscribed in the order given. The nearest known genes to rpoB are rplL and rplA,J,K, which code for ribosomal proteins, and which are transcribed in the same direction as the polymerase genes. It has been suggested that rpoBC may be distal elements of a larger operon including these ribosomal genes. To test this possibility we have cloned a segment of DNA, derived by endoR. HindIII digestion from the rpoBC-transducing bacteriophage lambdarifd18, in the replacement vector NMlambda761. The structure of the lambdarpoBC bacteriophages so produced is such that the inserted DNA can be transcribed from lambda promoters, allowing us to confirm that it carries intact rplL, rpoB, and rpoC genes. We have studied these bacteriophages as lysogens in rec+ and rec bacteria, and by infection of UV-irradiated bacterial strains in which lambda promoters are either repressed or active. The results indicate that the cloned DNA contains at most a very weak promoter for the above genes, in contrast to that present in the larger segment of bacterial DNA carried by lambdarifd18. We have in the same way cloned the adjacent bacterial HindIII-fragment of lambdarifd18 DNA, and have found that it displays vigorous autonomous expression of the tufB, rplA, and rplK genes. We conclude that rpoB and C are obligatorily co-transcribed with rplL, from a promoter located outside the DNA segment cloned in lambdarpoBC. We discuss the evidence for the existence of a regulatory site, rpoU, located between rplL and rpoB.

Bacterial Proteins↗
PubMed1978

Non-coordinate expression of the neighbouring genes rplL and rpoB,C of Escherichia coli.

The operon rpoB,C of Escherichia coli codes for the RNA polymerase subunits beta and beta'. rpoB procedes rpoC in the direction of transcription. The nearest characterised gene to rpoB on the chromosome is rplL, which codes for the ribosomal proteins L7/12. rplL appears to be transcribed in the same direction as rpoB,C, and it has been suggested that all three genes may lie in a single operon. The drug rifampicin induces increased production of beta and beta' in suitable strains of E. coli. We show here that alpha and sigma are also induced, whereas synthesis of L7/12 is not detectably affected.

DNA-Directed RNA Polymerases↗
PubMed1978

Over-synthesis and instability of sigma protein in a merodiploid strain of Escherichia coli.

We have used two different methods to study the rates of RNA polymerase subunit synthesis in haploid Escherichia coli K12, and a KLF10 rPOB, C+ merodiploid derivative, when grown in glucose-minimal medium at 37 degrees C. Our results indicate that the haploid strain produces beta, beta', alpha and sigma in the molar ratios 1.01:0.99: less than or equal to 2.90:0.26; and that all these subunits are reasonably stable during subsequent growth. The merodiploid produces alpha at the same rate as the haploid, beta and beta' at a 42% higher rate, and sigma at twice the rate. Some 40% of the newly synthesised beta and beta' is degraded within one hour; the residuum is as stable as in the haploid. Alpha is stable throughout. By contrast, sigma is subject to a marked and continuous turnover in the merodiploid. These results are discussed in terms of gene dosage and regulatory effects.

Chromosomes, Bacterial↗