Autogenous regulation of the RNA polymerase beta subunit of Escherichia coli occurs at the translational level in vivo (original) (raw)
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In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase
Proceedings of the National Academy of Sciences, 1982
Plasmids pNF1337 and pNF1341, which contain part of the LIO operon including the RNA polymerase 13-subunit gene, have been used as templates in vitro to investigate expression of the p-subunit gene. For these studies, the synthesis of the first dipeptide of the 13 subunit, fMet-Val, was measured instead of that of the entire protein. By using this dipeptide system, we studied the effects of RNA polymerase holoenzyme and L factor (nus A-gene product) on fMet-Val synthesis and compared the relative effects of the primary and secondary promoters in the L1O operon on expression of the 13-subunit gene. The results show that the inhibitory effect of RNA polymerase on P-subunit synthesis and the stimulatory effect of L factor occur before formation of the first dipeptide bond. In this in -vitro system, the secondary promoters account for about 50% of the total fMet-Val synthesized. Although the primary promoter is sensitive to guanosmne 5'diphosphate 3'-diphosphate in-vitro, the secondary promoters are not affected by this nucleotide.
Proceedings of the National Academy of Sciences, 1995
We present a simple, rapid procedure for reconstitution ofEscherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant a, ,B, (', and a7O subunits. Hexahistidine-tagged recombinant a subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant 13, 13', and cr70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, a-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with a(1-235), an a subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.
Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments
Proceedings of the National Academy of Sciences, 1995
The Escherichia coli rpoB gene, which codes for the 1342-residue j3 subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP 1 subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these p-subunit polypeptide fragments, when added with purified recombinant P', or, and a subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the 1f subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.
Journal of Molecular Biology, 1998
Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase a subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical a subunit containing the N-terminal domain of the E. coli a subunit and the CTD of the B. subtilis a subunit. Reconstitution of RNA polymerases containing this chimerical a subunit, the E. coli b and b H subunits, and the vegetative s factor from either E. coli (s 70) or B. subtilis (s A), generated hybrid enzymes that were responsive to protein p4 and ef®ciently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis a-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.
A beta Subunit Mutation Disrupting the Catalytic Function of Escherichia coli RNA Polymerase
Proceedings of The National Academy of Sciences, 1991
The substitution of the evolutionarily conserved Glu-813 for lysine in the (3 subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP. In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate. Both defects could be compensated by raising the ribonucleoside triphosphate concentration. In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex. A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.
Altered promoter recognition by mutant forms of the ?70 subunit of Escherichia coli RNA polymerase*1
J Mol Biol, 1989
We have systematically assayed the in viva promoter recognition properties of 13 mutations in rpoD, the gene that encodes the 0" subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of c" and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of cr", which is similar to the helix-turnhelix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the-35 region, and that a sequence internal to a", in a region which is highly conserved among Q factors, recognizes the-10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the-35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effects on promoter recognition.
MGG Molecular & General Genetics, 1980
As has been shown previously, RNA polymerase fl' subunit ts-mutation rpoC1 results in an overproduction of RNA polymerase tiff" subunits at nonpermissive temperature. The mutant enzyme shows low activity in vitro and a sedimentation coefficient 9S which is characteristic of immature core polymerase. In this paper we describe a mutation designated oprl which suppresses RNA polymerase ~fi' subunit overproduction. The mutation was found among Ts + revertants of the Ts double mutant carrying a rpoC1 mutation and a rif-r rpoB251 mutation. Oprl is closely linked to the original rpo mutations and shows complete trans-dominance. Although oprl seems to affect RNA polymerase, it does not suppress the accumulation of immature 9S RNA polymerase and does not restore the activity of the RpoC1 mutant enzyme. This and other results of a comparison of strains carrying different combinations of rpoC1, rpoB251 and oprl mutations suggest that neither inhibition of total RNA and protein synthesis, nor the low RNA polymerase activity in vitro, nor the apparent defects in enzyme maturation, nor the enzyme degradation observed at 42 ° C are responsible for the fi~' overproduction in RpoC1 strains.
Journal of Bacteriology, 2000
Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the  and  subunits and the downstream DNA duplex. on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from on June 23, 2015 by guest http://jb.asm.org/ Downloaded from