Conformation of fork junction DNA in a complex with Escherichia coli RNA polymerase

Escherichia coli RNA polymerase is able to bind fork junction DNA containing a conserved -10 promoter element in a sequence-specific manner, and it is believed that polymerase-fork junction DNA interaction mimics those between the enzyme and the promoter DNA in the open complex. In this report we determined the conformation of polymerase-bound fork junction DNA in solution. A series of distances between sites in the fork junction DNA in complex with polymerase were determined using luminescence and fluorescence resonance energy transfer. A series of fork junction DNAs were prepared containing the luminescent or fluorescent donor probe at the upstream or at the downstream end of the fork DNA and acceptor probes at nine positions within the fork junction DNA. The measured distances were compared with analogous distances in a model reference DNA duplex, and the observed distance differences were used to build a model of the fork junction DNA in a complex with the polymerase. The obtained model revealed an insignificant perturbation of the duplex part of the fork DNA in a complex with the polymerase whereas a sharp kink of DNA was observed at the ds/ss DNA boundary of the fork junction DNA.     

DNA-dependent RNA polymerase of Escherichia coli induces bending or an increased flexibility of DNA by specific complex formation.

Escherichia coli RNA polymerase is shown to induce bending or an increased flexibility of the promoter DNA. This is a specific effect of holoenzyme (core enzyme and sigma-factor). The centre of the flexibility is 3 bp upstream of the initiation point of RNA synthesis. This flexibility or bending is maintained during RNA synthesis by core enzyme.     

Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.     

Escherichia coli RNA polymerase binding to a DNA terminus prevents formation of a closed promoter complex

A 302 bp DNA fragment and a 113 bp subfragment of the former, both containing the fd gene VIII promoter (P VIII), were found to exhibit temperature-dependent differential behaviour in RNA chain initiation from P VIII. At 37 degrees C no significant differences were observed, while at 17 degrees C chain initiation was strongly suppressed only with the 113 bp fragment. This phenomenon depended on the presence of the (blunt) DNA terminus upstream from P VIII (position -70). Footprinting revealed that at 17 degrees C RNA polymerase was bound to this DNA fragment in a different mode. Contacts were observed only upstream from position -25. On the contrary, at 37 degrees C only the promoter complex footprint was visible. These results indicate that at 17 degrees C formation of the non-initiating complex is more favourable than formation of the promoter complex (which is closed at 17 degrees C; Hofer, B., Müller, D. and K?ster, H. (1985) Nucleic Acids Res. 13, 5995-6013) and that formation of both complexes is mutually exclusive. No footprints of RNA polymerase were observed at other DNA termini. This indicates a sequence-specificity for the interaction at the terminus of the 113 bp fragment. The footprint pattern, together with features of the DNA sequence, suggests that the contacts involved in this interaction are similar to those promoter contacts formed upstream from position -20 and that DNA without a -10 region can be specifically recognized by RNA polymerase.     

Evidence for specific control of RNA polymerase synthesis in Escherichia coli

Studies on the synthesis of RNA polymerase in E. coli rif mutants containing both sensitive and resistant RNA polymerase molecules show that the synthesis of E. coli RNA polymerase is under a specific and active control system.     

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli

The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str. Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation. Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T. V. Bhuvaneswari, B. G. Turgeon, and W. D. Bauer, Plant Physiol. 66:1027-1031, 1980). Profiles of nodulation frequency at different locations on the root were not affected by the age of the R. japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation. Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection. Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses. Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin. Suspensions of R. japonicum in water were found to retain their viability and infectivity.     

Characterization of an RNA "binding site" for a specific ribosomal protein of Escherichia coli

Summary A portion of the 16S ribosomal RNA that binds specifically to, and is protected from nucleolytic attack by, ribosomal protein S4 has been characterized in terms of its partial primary structure. The specific RNA (S4aR) in question comprises slightly more than onefourth of the full 16S molecule, and appears to be located (at least in part) in the 5-proximal half of the molecule. The functional significance of S4aR is discussed.     

Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu

The products of nitrous acid mediated-deamination of Phe-tRNAPhe from E. coli were analyzed and their capability to interact with elongation factor Tu from E. coli was investigated. Thin-layer chromatography as well as HPLC analysis revealed the existence of at least two deamination products, 3-phenyl-lactyl-tRNAPhe and cinnamyl-tRNAPhe. It could be shown that the aminoacyl-tRNA analogues were active in the formation of the ternary complex with EF-Tu X GTP, although with a lower efficiency than native Phe-tRNAPhe. For both modified acyl-tRNAs the dissociation constant was determined to be 3 X 10(-5) M.