Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs.

The interaction of Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) with restriction fragments obtained from various E. coli related DNAs was studied in vitro. The DNAs investigated included several coliphage genomes (T5, lambda, T7, fd) and plasmid DNAs (pML 21, pSC101). By using the nitrocellulose filter binding of the enzyme-DNA complexes, fragment-specific relative rates of complex formation as well as complex stabilities were determined. Promoter-specific relative rates of polymerase binding were derived from fragment-specific rates by taking into account the number of major binding sites for RNA polymerase within several DNAs. Estimates of the stability of complexes formed between some major binding sites and the enzyme were obtained by studying the rate of complex decay. Both characteristics--rate of complex formation and rate of decay--varied widely and independently of each other. The promoters reacting most efficiently with E. coli RNA polymerase were found in the early region of coliphage T5 whereas some promoters in pML 21, or for example, the lambda promoter PI, belong to signals binding the enzyme most slowly. Based on the second-order rate constant determined for the interaction of E. coli RNA polymerase with promoters of phage fd, the fastest promoters characterized so far reacted with rates in the order of 10(8) M-1s-1. The hierarchy of promoters established here is of interest from the viewpoint that promoter strength correlates with the rate of polymerase binding. Among the promoters studied here this rate spans a range of 2 orders of magnitude.     

New Small Polypeptides Associated with DNA-Dependent RNA Polymerase of Escherichia coli after Infection with Bacteriophage T4

Four new small polypeptides are associated with DNA-dependent RNA polymerase from E. coli after infection with T4 phage. The new polypeptides are easily detected in RNA polymerase from E. coli cells labeled with amino acids after phage infection. Their molecular weights range from 10,000 to 22,000, as detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All four polypeptides are found after infection with either wild-type T4 phage or T4 early amber mutants in genes 44, 42, 47, and 46. None of the polypeptides is labeled significantly before 5 min after infection at 30 degrees . When two maturation-defective amber mutants in gene 55 of T4 phage are used for infection, a polypeptide with a molecular weight of 22,000 is absent. When a maturation-defective amber mutant in gene 33 of T4 phage is used, another small protein is absent.     

Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7.

Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl-transferase, EC2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before RNA initiation site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Pribnow box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.     

Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA.

Electron microscopy was used to study the formation of random complexes between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 3 degrees to the promoter-containing (Hinf)1100 fragment of the same DNA. The value of the Ka of random binding is about 3 times 10(4)M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37 degrees. If dilution is rapid and occurs in a single step, or if incubation extends beyond 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 10(8)M-1). Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought. However, great caution is required in assessing the extent of random binding where damage to the enzyme may occur. When RNApolymerase holoenzyme is incubated at 0 degrees with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37 degrees, although the highly stable "open" promoter complex is not formed under these conditions. However, the extent of binding is reduced as compared to promoter complexes formed at 37 degrees. This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical "closed"complexes formed between RNA polymerase and duplex DNA.     

Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli.

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.