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)

We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.
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PMID:Antitermination of characterized transcriptional terminators by the Escherichia coli rrnG leader region. 218 97

The frequency of transcription of the ribosomal protein and RNA polymerase gene segments of the rplKAJL-rpoBC gene cluster was measured for Escherichia coli K-12 strains carrying mutations in the genes for transcriptional termination factors. The results of our study suggest that Rho increases and that both NusA and the product of sfrB decrease termination frequency in the rplL-rpoB intercistronic region.
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PMID:Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region. 243 1

The complete nucleotide sequence of a 3541-base pairs (bp) DNA fragment from Bacillus stearothermophilus able to complement an Escherichia coli glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mutant (gapD-) has been determined. The B. stearothermophilus gap gene consists of a 1005-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Upstream from the start codon is a strong Shine-Dalgarno sequence typical of Gram-positive bacteria. Only one putative RNA polymerase recognition signal (-35 and -10 regions) is found 1153 bp upstream from the ATG start codon. In vivo utilization of this signal is in agreement with the study of gene expression from different subclones of the original fragment. 57 bp downstream from the TAA stop codon is a structure resembling Rho-independent transcription termination signals. Although B. stearothermophilus GAPDH-coding gene is highly expressed in E. coli, it contains several rare codons for E. coli. The predicted amino acid sequence of the GAPDH enzyme presents several differences with the amino acid sequence previously determined from the protein and is in better agreement with published crystallographic data.
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PMID:Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli. 265 7

We examined the in vitro concentration dependence of the effects of Nus A on transcription termination and pausing to determine if Nus A affects both pausing and termination in vitro by binding to a single site on the transcription complex. Nus A was shown to cause maximal increases of pausing at a concentration approximately equimolar to RNA polymerase. However, the effects of Nus A on termination require much higher Nus A concentrations than are required for pausing. It is therefore likely that the effects of Nus A on pausing and termination result from the binding of Nus A to different sites on the transcription complex. Since proteins that probably bind RNA nonspecifically were also shown to strongly reduce termination at a Rho-dependent terminator, Nus A may decrease Rho-dependent termination by binding nonspecifically to RNA. This proposal is consistent with most of the available data on the in vitro effects of Nus A and provides a mechanistic basis for previously unexplained details of Nus A caused decreases in Rho-dependent termination. We further speculate that most or all of the in vivo roles of Nus A may involve the enhancement of pausing.
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PMID:Nus A protein affects transcriptional pausing and termination in vitro by binding to different sites on the transcription complex. 284 44

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.
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PMID:Sequence of the Bacillus subtilis glutamine synthetase gene region. 290 11

Bacteriophage lambda N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.
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PMID:Evidence that ribosomal protein S10 itself is a cellular component necessary for transcription antitermination by phage lambda N protein. 298 61

The structural gene for L-lactate dehydrogenase (LDH; EC 1.1.1.27) from Bacillus stearothermophilus NCA 1503 has been cloned in Escherichia coli and its complete nucleotide sequence determined. The predicted amino acid (aa) sequence of the LDH enzyme agrees with the previously determined aa sequence except to three positions: aa 125 and 126, Ser-Glu, are inverted whilst His at position 130 has been replaced by Ser in our sequence. The lct gene consists of an open reading frame (ORF) commencing from the ATG start codon of 951 bp followed by a TGA stop codon. Upstream from the start codon is a strong (delta G = -14.4 kcal) Shine-Dalgarno (SD) sequence, a feature typical of Gram-positive ribosome binding sites. Putative RNA polymerase recognition signals (-35 and -10 regions) have been identified upstream from the lct structural gene but there are no structures resembling Rho-independent transcription termination signals downstream from the TGA stop codon. Two further ORFs, preceded by SD sequences, are present downstream from the lct gene. Thus the lct gene may constitute the first gene of an operon. Subclones of the lct gene have been constructed in the expression plasmid pKK223-3 and the LDH enzyme produced in soluble form at levels of up to 36% of the E. coli soluble cell protein.
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PMID:Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene. 302 26

The highly defective rho-15 mutant of Escherichia coli produces plasmid DNA that is 22% less negatively supercoiled than DNA from an isogenic wild-type strain (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We extended our measurements of plasmid superhelicity to additional rho mutants and to strains containing mutations that suppress rho transcription termination defects; the suppressor mutations were in the rpoB and the rho genes. The superhelicity of plasmid DNA was reduced by 11 and 10%, respectively, in the rho-702 and rho-201 mutants, both of which are less defective in Rho-mediated transcription termination than rho-15. Plasmid superhelicity was restored in all the suppressed rho mutants; in one rpoB mutant, plasmid DNA was even more negatively supercoiled than in rpoB+ cells, whether in a rho+ or rho mutant background. Suppression of rho mutants enabled them to maintain plasmids that could not be maintained in the mutants in the absence of the suppressor mutations. The results indicate that in addition to DNA gyrase, topoisomerase I, and Rho, RNA polymerase is also a determinant of DNA superhelicity, and its effect is modified by the Rho protein. We propose that Rho may increase the degree of DNA unwinding by the transcription complex, possibly at transcription termination sites.
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PMID:Regulation of DNA superhelicity by rpoB mutations that suppress defective Rho-mediated transcription termination in Escherichia coli. 304 90

Rifampicin-resistant (Rifr) mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have examined the effect of each of the 17 sequenced Rifr mutations in our collection on transcription termination. The effect of each Rifr mutation was measured at three types of terminators: simple terminators requiring only RNA polymerase to terminate in vitro, and complex terminators requiring either Rho or Tau for in-vitro termination. Almost every Rifr allele examined (14/17) affected readthrough at one or more of these terminators. We found that mutations with similar termination phenotypes were clustered suggesting functional specialization within the region of rpoB defined by the Rifr mutations. The interaction of the Rifr mutations with the defective rho15 allele was also investigated. Only two Rifr mutations suppress the termination defect of rho15 strains. We discuss models to explain how this region of the beta polypeptide might be involved in the process of transcription termination.
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PMID:Characterization of the termination phenotypes of rifampicin-resistant mutants. 305 Jan 23

Expression plasmids containing the E. coli lacZ coding region preceded by a set of different ribosome-binding sites and put under transcriptional control of the leftward promoter of phage lambda (PL) were used to study the synthesis of lacZ mRNA. In a normal host the steady state level of full-length lacZ mRNA varied 100-fold with the different synthesis levels of beta-galactosidase, whereas in a host expressing the antitermination protein N of phage lambda, all vectors synthesized the same amount of full-length lacZ mRNA, while maintaining the differences in beta-galactosidase expression. We present evidence for a causal relationship between the rate of ribosome loading and the continuation of transcription across the lacZ gene. We suggest that extended spacing between the RNA polymerase and the elongating ribosome causes transcriptional polarity by increasing the extent of premature termination. The conditional character of the termination event can best be explained by invoking termination factor Rho.
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PMID:Inefficient translation initiation causes premature transcription termination in the lacZ gene. 308 Dec 64


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