Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system. The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB. Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination. The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination. The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.
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PMID:Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components. 843 Jan 11

The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.
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PMID:Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins. 853 55

A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues.
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PMID:Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus. 855 81

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.
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PMID:Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex. 866 50

Escherichia coli RNA polymerase is composed of four different subunits, alpha (present in two copies), beta, beta' and sigma. Among these, the beta' polypeptide shares nine conserved regions with the largest subunits of eukaryotic RNA polymerases, but its role is poorly understood. We isolated novel mutations in a plasmid-borne copy of rpoC, which encodes beta', as dominant suppressors of two temperature-sensitive nusA alleles. All 20 suppressors of nusA11 (single missense mutation) isolated had either of two specific substitutions: Lys for Glu-402 (rpoC10) and Thr for Ala-904 (rpoC111) in the beta' subunit. In vivo and in vitro transcription assays revealed that the rpoC10 allele of beta' participates in Rho-dependent transcription termination. On the other hand, of 20 suppressors of nusA134 (deletion of C-terminal one-third) scattered at 18 distinct sites, 16 were assigned to one of six conserved regions C-I. These results suggested that the conserved domains of the beta' subunit of E. coli RNA polymerase are involved in transcript termination or interaction with termination factor(s).
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PMID:Localization of nusA-suppressing amino acid substitutions in the conserved regions of the beta' subunit of Escherichia coli RNA polymerase. 875 1

We previously found that nusD-type mutations in Escherichia coli transcription termination factor Rho enhance in vitro transcription termination at four points within the lambdacro gene. Here we show that the early termination points are part of one Rho-dependent termination site, tRE, with properties like those of previously characterized Rho-dependent sites lamda tR1 and trpt'. The early termination points are all RNA polymerase pause sites, and by deletion analysis and oligonucleotide blocking experiments, a common 5' Rho entry site for the early termination points (rutE) is identified. We show that both Rho026 and Rho+ can use rutE as an entry point for termination, but that Rho026 is more efficient in releasing the nascent RNA at tRE. The RNA-dependent ATPase activities of wild-type and mutant Rhos are similar, as are their abilities to bind free RNA and to use (rC)10 oligomers for ATPase activation. We therefore suggest that Rho-RNA polymerase interactions that define the site of RNA 3' end formation are altered in NusD Rho mutants. NusD Rho mutants are less dependent on, but still responsive to, the transcription termination factor NusG. However, addition of NusG to in vitro termination assays allows Rho+ to terminate more efficiently at tRE. These results suggest that NusG aids in the 3' end formation process. The decreased dependence on NusG for termination by the mutant Rhos in vitro provides an explanation for poorer lambda growth in rho(nusD) cells by interference with lamdaN-mediated antitermination at Rho-dependent sites.
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PMID:The mechanism of early transcription termination by Rho026. 875 98

The interaction of Rho and the antibiotic bicyclomycin was probed using in vitro transcription termination reactions, poly(C) binding assays, limited tryptic digestions, and the bicyclomycin inhibition kinetics of ATPase activity in the presence of poly(dC) and ribo(C)10. The approximate I50 value for the bicyclomycin inhibition of transcription termination at Rho-dependent sites within a modified trp operon template was 5 microM. At antibiotic concentrations near the I50 value, bicyclomycin inhibition of Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts whose size was midway between the Rho-dependent transcripts and the readthrough transcripts. Bicyclomycin did not inhibit poly(C) binding to Rho. In the presence of poly(dC), bicyclomycin showed a reversible mixed inhibition of the ribo(C)10-stimulated ATPase activity. The extrapolated Ki for bicyclomycin was 2.8 microM without ribo(C)10 and increased to 26 microM in the presence of ribo(C)10. Correspondingly, the Km(app) for ribo(C)10 without bicyclomycin was 0.8 microM and with bicyclomycin was 5 microM at infinite inhibitor concentration. The data suggested that the antibiotic binds to Rho, influencing the secondary RNA binding (tracking) site on Rho and slows the tracking of Rho toward the bound RNA polymerase.
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PMID:The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho. 881 Mar 2

Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.
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PMID:Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus. 899 24

Ribosomal RNA (rRNA) is elongated twice as fast as mRNA in vivo due to the presence of antitermination sequences in the 5' part of the rRNA transcripts. A number of Nus factors bind to RNA polymerase at the antitermination sites and help confer resistance to Rho-dependent termination of transcription. In this paper, the effects of the nusAcs10 allele on the elongation rate of both mRNA and antiterminated RNA were investigated. The results indicate that NusA is required to achieve a high elongation rate of RNA chains carrying the ribosomal antitermination boxA and that antitermination is defective when the rate of transcription elongation is decreased by the nusAcs10 allele. Furthermore, the nusAcs10 allele had no significant effects on the elongation rate of normal lacZ mRNA during steady state growth, but it abolished the inhibition of lacZ mRNA elongation by guanosine 3',5'-bis(diphosphate) (ppGpp). These results suggest that NusA is the component of the transcription elongation complex required for inhibition of mRNA elongation by ppGpp.
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PMID:NusA is required for ribosomal antitermination and for modulation of the transcription elongation rate of both antiterminated RNA and mRNA. 913 68

The association of the transcriptional antitermination protein N of bacteriophage lambda with Escherichia coli RNA polymerase depends on nut site RNA (boxA + boxB) in the nascent transcript and the host protein, NusA. This ribonucleoprotein complex can transcribe through Rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut. For antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins NusB, NusG, and ribosomal protein S10. Here, we show that the assembly of NusB, NusG, and S10 onto the core complex involves nucleotides 2-7 of lambda boxA (CGCUCUUACACA) and is a fully cooperative process that depends on the presence of all three proteins. This assembly of NusB, NusG, and S10 also requires the carboxyl-terminal region (amino acids 73-107) of N, which interacts directly with RNA polymerase. NusB and S10 assemble in the absence of NusG when lambda boxA is altered at nucleotides 8 and 9 to create a consensus version of boxA (CGCUCUUUAACA). These experiments suggest that multiple protein-protein and protein-RNA interactions are required to convert a core antitermination complex into a complete complex.
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PMID:Involvement of boxA nucleotides in the formation of a stable ribonucleoprotein complex containing the bacteriophage lambda N protein. 946 9


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