Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation

RNA polymerase interactions and elongation rate

We show that non-steric molecular interactions between RNA polymerase (RNAP) motors that move simultaneously on the same DNA track determine strongly the kinetics of transcription elongation. With a focus on the role of collisions and cooperation, we introduce a stochastic model that allows for the exact analytical computation of the stationary properties of transcription elongation as a function of RNAP density, their interaction strength, nucleoside triphosphate concentration, and rate of pyrophosphate release. Cooperative pushing, i.e., an enhancement of the average RNAP velocity and elongation rate, arises due to stochastic pushing. This cooperative effect cannot be explained by steric hindrance alone but requires a sufficiently strong molecular repulsion. It disappears beyond a critical RNAP density, above which jamming due to collisions takes over. For strong stochastic blocking the cooperative pushing is suppressed at low RNAP densities, but a reappears at higher densities.Comment: 26 pages, 6 figure

A comparative study of DNA-dependent RNA polymerases from rat ascites hepatoma cell nuclei and from rat liver nuclei

DNA-dependent RNA polymerases (EC 2.7.7.6) were extracted and partially purified form the nuclei of rat ascites hepatoma cells (AH-130) induced by 4-dimethylaminoazobenzene. The patterns of RNA synthesis and the properties of these enzymes were compared with enzymes from the nuclei of rat liver. The specific activity of RNA polymerase in the homogenate from the nuclei of AH-130 cells was the same as normal rat liver nuclei. RNA polymerase was solubilized from the homogenate at high ionic strength and separated into two forms by DEAE-Sephadex column chromatography. Enzymatic characterization showed that these enzymes corresponded to RNA polymerase I and II. RNA polymerase I more effectively transcribed native DNA than denatured DNA at low salt concentration, but at high salt concentration RNA polymerase I effectively transcribed denatured DNA. RNA polymerase II more effectively transcribed denatured DNA. In AH-130 cells the activity of RNA polymerase I was 4 to 5 times higher than RNA polymerase II, and in rat liver the activity of RNA polymerase I was 1.5 to 2 times higher than RNA polymerase II. The activity of RNA polymerase I in AH-130 cells may have increased by induction.</p

Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase

Transcription in all living organisms is accomplished by multi-subunit RNA polymerases (msRNAPs). msRNAPs are highly conserved in evolution and invariably share a B400 kDa five-subunit catalytic core. Here we characterize a hypothetical B100 kDa single-chain protein, YonO, encoded by the SPb prophage of Bacillus subtilis. YonO shares very distant homology with msRNAPs, but no homology with single-subunit polymerases. We show that despite homology to only a few amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive DNA-dependent RNA polymerase. We demonstrate that YonO is a bona fide RNAP of the SPb bacteriophage that specifically transcribes its late genes, and thus represents a novel type of bacteriophage RNAPs. YonO and related proteins present in various bacteria and bacteriophages have diverged from msRNAPs before the Last Universal Common Ancestor, and, thus, may resemble the single-subunit ancestor of all msRNAPs

alpha-helix E of Spo0A is required for sigma(A)- but not for sigma(H)-dependent promoter activation in Bacillus subtilis

At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by sigma(A)-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor sigma(H). Previous genetic analyses have identified specific amino acids in alpha-helix E of Spo0A that are important for activation of Spo0A-dependent, sigma(A)-dependent promoters. However, these amino acids are not required for activation of the sigma(H)-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in alpha-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the sigma(A)-dependent promoters. Based on the effects of a deletion mutation, we suggest that alpha-helix E in Spo0A is not directly involved in interaction with sigma(H)-RNA polymerase

Rescue of Synthetic Genomic RNA Analogs of Rabies Virus by Plasmid-Encoded Proteins

Proteins eolirely expressed from cDNA wen used to rescue synthetic RNA genome analogs into infectious defective particles or rabies virus (RV). Synthetic negative-stranded RNAs coßtalning 3&apos; · and S&apos;-terminal RV sequences and tnlßscriptional signal sequences wen transcribed (rom plasmids transfeded into cells expressing 1&apos;7 RNA polymerase (rom recombinant vaccinia virus. After simultaneous expression or RV N, P, and L proteiDS (rom plasmids containing a T7 RNA polymerase promoter, tbe synthetic genomes wen encapsidated. replicated, and transcribed by tbe RV polymerase proteiDS. Insertion or the bac1erial chloramphenicol acetyUransferase gene or l3·galactosidase (IacZ) gene between the 3 &apos; and 5 &apos; termini containing transcriptional signal sequenees resulted in transcription of mRNAs and expression of ehloramphenlco

The largest subunit of human RNA polymerase III is closely related to the largest subunit of yeast and trypanosome RNA polymerase III

In both yeast and mammalian systems, considerable progress has been made toward the characterization of the transcription factors required for transcription by RNA polymerase III. However, whereas in yeast all of the RNA polymerase III subunits have been cloned, relatively little is known about the enzyme itself in higher eukaryotes. For example, no higher eukaryotic sequence corresponding to the largest RNA polymerase III subunit is available. Here we describe the isolation of cDNAs that encode the largest subunit of human RNA polymerase III, as suggested by the observations that (1) antibodies directed against the cloned protein immunoprecipitate an active enzyme whose sensitivity to different concentrations of alpha-amanitin is that expected for human RNA polymerase III; and (2) depletion of transcription extracts with the same antibodies results in inhibition of transcription from an RNA polymerase III, but not from an RNA polymerase II, promoter. Sequence comparisons reveal that regions conserved in the RNA polymerase I, II, and III largest subunits characterized so far are also conserved in the human RNA polymerase III sequence, and thus probably perform similar functions for the human RNA polymerase III enzyme