Visualization of single molecules of RNA polymerase sliding along DNA.

Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

Properties of nerve growth factor from the venom of Bothrops atrox.

A glycoprotein fraction islated in poor yield (approx. 0.04%) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-reactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against nerve growth factor from mouse had little effect on any of these preparations.

The effect of a nested set of C-terminal substituted deletions on the function of the alpha subunit of Escherichia coli RNA polymerase.

A genetic screen was devised to obtain plasmid-borne rpoA alleles exhibiting partial or no complementation of the chromosomal Escherichia coli rpoA341 allele responsible for a pleiotropic phenotype. Nine of the ten mutants obtained carried single base pair deletions within the 3' end of rpoA resulting in frameshifting into a 72 codon +1 orf extending from within codon L262 and terminating 16 bp downstream of the rpoA reading frame. These frameshifts give rise to a set of substituted alpha deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the alpha region from the C-terminus (residue 329) to various points between 272 > 319. The in vivo properties of this nested set of nine C-terminal-substituted derivatives of the alpha subunit of RNA polymerase have been assessed in terms of their assembly and transcriptional proficiency. The results indicate: (i) replacement of as much as 42 C-terminal residues of the alpha subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA112(Ts); (ii) the extreme C-terminal Orf region, like that of alpha itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters.

Involvement of the Escherichia coli RNA polymerase alpha subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins.

Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.

'Stop-codon-specific' restriction endonucleases: their use in mapping and gene manipulation.

Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG and are commonly either 4 or 6 bp in length. Interestingly, these restriction targets do not occur at the frequency expected on the basis of base composition and size. For example, the tetranucleotide MaeI recognition sequence (CTAG) occurs considerably less commonly (5-8-fold) in the genome of Escherichia coli (and many other eubacteria) than expected from mononucleotide frequencies. This surprising rarity is particularly evident in protein-encoding genes and is largely dictated by codon usage. Thus, amber (TAG) nonsense mutations frequently give rise to novel MaeI (CTAG) sites which are unique within a translated region. Such amber/MaeI sites, whether arising spontaneously or created in vitro by site-directed mutagenesis, act as a useful physical marker for the presence of the nonsense mutation and are a convenient startpoint for a range of diverse procedures. These features provide a useful supplement to protein engineering methods which use nonsense suppression to mediate amino acid replacements.

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.

Relaxed mutants of Escherichia coli RNA polymerase.

Unusual guanosine nucleotides synthesised during amino acid or energy source starvation are thought to be the effectors of the stringent response. In vitro experiments suggest that the magic spot compounds alter transcription specificity of RNA polymerase by binding to the enzyme. However, there is no good in vivo evidence for such an interaction. We define sites on the beta-subunit of RNA polymerase which, when altered, yield E.coli mutants apparently insensitive to the presence of ppGpp.

The patterns of extracellular protein formation by spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus.

Spontaneously-occurring rifampicin-resistant mutants of Staphylococcus aureus were isolated on 4% (w/v) Tryptone Soya Agar containing 4 and 40 times the m.i.c. for rifampicin. A number of colonies were selected at each rifampicin concentration and were grown aerobically in 3% (w/v) Tryptone Soya Broth for 24 h at 37 degrees C. In the case of S. aureus RN4220 all the mutants grew to bacterial densities up to approximately 1.7 times more than the parent organism. The corresponding levels of extracellular protein secretion varied over a 5-fold range, all the mutants being less productive than the parent. By contrast, mutants of the wild-type Wood 46 strain achieved bacterial densities of only 45-83% that of the parent whilst exoprotein secretion showed a smaller 1.7-fold variation. However, widely-differing patterns of exoproteins were revealed by SDS-polyacrylamide gel electrophoresis of the parent and mutant organisms of both strains.

Direct measurement of absolute suppressor efficiency.

We have devised a system for measuring the degree of translational readthrough past a nonsense mutation which is based upon the quantitation of the two translation products, the suppressed polypeptide and the nonsense fragment. The absolute efficiency of four different amber suppressors (Su1, Su2, Su3, and Su7) has been determined at two unique amber sites in the structural gene for the beta subunit of Escherichia coli RNA polymerase.

Surgical audit in a district general hospital: a stimulus for improving patient care.

A computerised audit system was used to monitor clinical and administrative management of surgical patients in a District General Hospital. In one year (1985) there were 1,060 discharges from 27 beds (39.3 patients/bed/year). Operative treatment was required in 652 patients but 408 patients did not undergo surgery. Of this latter group the majority were admitted with abdominal pain (179 patients) and a final diagnosis was made in only 76 patients. The majority of patients were discharged within one week of admission, but 94 patients (8.9%) were admitted for more than three weeks. Of this group 42.5% could not be discharged because of poor home and social circumstances. The results of this study suggest that management could be improved without compromising patient care, in particular by providing adequate facilities for day care surgery and by improving social and geriatric support services. Reductions in staff or bed numbers without providing such improvements will lead to a significant reduction in patient care.

Duration of symptoms and spread of colorectal cancer: a short history does not mean early disease.

The 5-year survival rates for colorectal cancer are generally lower in the UK than other European countries. In an attempt to improve prognosis, central government has stipulated that patients with suspicious symptoms ought to be seen within 2 weeks of referral from a primary care physician. In order to evaluate whether symptom duration affects stage at presentation of colorectal cancer, a retrospective analysis of all patients presenting over a 2-year period to a large district general hospital was performed. There was no significant difference (P = 0.885) in Dukes' staging in patients with symptoms lasting less or more than 6 months. Though seeing patients with symptoms suspicious of colorectal cancer in specialist out-patient clinics within 2 weeks of presentation to the primary care physician would probably reduce the number of patients presenting as an emergency, it is unlikely to improve prognosis. Thus funds diverted towards the 2-week wait are probably best utilised for other procedures such as colonoscopy and for improving care once the diagnosis of cancer has been made. Diagnosis of colorectal cancer at an earlier stage is best achieved by screening of the population.

In vivo cloning of a carboxy-terminal rpoB allele which confers altered transcriptional properties.

A 3'-terminal mutation of the gene encoding the beta subunit of Escherichia coli RNA polymerase was isolated using an in vivo polA(Ts) technique. Cloning of the allele was monitored by virtue of the fact that the deletion delta(rpoB)1570-1 resulted in an altered-size restriction fragment. DNA sequencing confirmed the predicted nature and location of the mutation: delta(rpoB)1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967-1028. The phenotype conferred by delta(rpoB)1570-1 is discussed with respect to conserved domains within the beta polypeptide.

Identification of an amber fragment of the beta subunit of Escherichia coli RNA polymerase: a yardstick for measuring controls on RNA polymerase subunit synthesis.

An amber fragment of the beta subunit of Escherichia coli RNA polymerase has been recovered from strains carrying the rpoB12 amber mutation, indicating that the B12 mutation resides in the structural gene for the beta subunit. The fragment is readily assayed and can be used to determine the degree of expression of a single rpoB cistron in strains haploid or diploid for this region. These studies confirm that the bacterial mechanism, which can compensate for reduced translation of the beta message, operates by the co-ordinate induction of rpoB and rpoC. Furthermore, I show that rpo control depends upon cistron(s) located on the F' factor, KLF10, whose product(s) can act negatively in trans on rpoBC expression.

Escherichia coli rpoA mutation which impairs transcription of positively regulated systems.

The rpoA341 (phs) mutation of Escherichia coli results in decreased expression of several positively regulated operons and has been mapped to within or very near the rpoA gene encoding the alpha subunit of RNA polymerase. We have shown that plasmid-directed synthesis of the wild-type alpha subunit can complement the defective phenotypes associated with this mutation consistent with its proposed location within rpoA. This mutation was mapped by marker rescue to within a 182bp region near the 3' end of rpoA and was subsequently transferred to a plasmid by recombination in vivo. DNA sequence analysis revealed that the RpoA341 phenotype was the result of the substitution of lysine 271 by glutamate within the alpha polypeptide. We discuss this result in relation to our current understanding of the functional organization of the alpha subunit.

Bacterial RNA polymerases: structural and functional relationships.

The essential role of DNA-dependent RNA polymerases in gene expression and the fact that the multimeric species are highly conserved throughout nature makes these enzymes a particular fascinating area of study. Here we shall review the conservation of structures and their relationship to function, especially in the multimeric eubacterial RNA polymerases, paying particular attention to the ß core subunit and to recent studies of σ-factors of both the σ (70) and σ (54) families. We shall conclude with a brief consideration of phage-encoded RNA polymerases and phage-mediated modification of the host enzyme, and of the evolution of RNA-synthesising enzymes.

Function in vivo of separate segments of the beta subunit of Escherichia coli RNA polymerase.

BACKGROUND: Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed beta and beta', are conserved throughout nature. The beta subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the beta subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the beta subunit of RNA polymerase. To this end, the beta structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector. RESULTS: Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the beta polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo. CONCLUSIONS: These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of beta may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF-Tu in stringent control.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. IV. Structure-function correlates.

We have isolated a set of strains that synthesise 331 potential variants of E. coli DNA-dependent RNA polymerase making use of nonsense suppression of amber mutations in the beta structural gene; rpoB. Translational mapping, together with the effect of known amino acid substitutions, has allowed us to locate sites on the beta polypeptide involved in transcription termination, stringent response and resistance to the antibiotic rifampicin. In general, the C-terminal quarter appears to be less affected by such single amino acid exchanges than the rest of beta. These studies permit for the first time structure-function correlates for the beta subunit of RNA polymerase.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VI. A redundant region in the beta polypeptide.

A single species of RNA polymerase is responsible for the transcription of genetic material in Escherichia coli; and, each component of this essential enzyme is encoded by a single gene. Nevertheless, we report the characterisation of a viable deletion mutant lacking about 165 bp in the region coding for residues 965 to 1143 of the beta subunit of this enzyme. The foreshortened beta polypeptide appears to function as normal, suggesting that this region is, in fact, non-essential with regard to the usual functions of E. coli RNA polymerase.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. I. The effect of known, single amino acid substitutions in an essential protein.

The use of five different nonsense suppressors, in conjunction with a collection of 95 independent, spontaneously-occurring amber mutants affecting expression of rpoB, allows the generation of a maximum of 475 potential variants of the beta subunit of E. coli RNA polymerase, each carrying a known amino acid substitution at a particular site. The effect of these amino acid exchanges has been investigated in vivo. A significant majority (363/475) of substitutions lead to cellular death and altered properties--temperature sensitivity, apparently altered transcription termination and a changed stringent response--indicating that RNA polymerase function (unlike that of dispensable proteins) is extremely sensitive to such single site changes.