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 T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E. Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity. We also found that T7 RNA polymerase terminates within the 5S rRNA gene.
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PMID:The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli. 171 77

RNase E is a major endonucleolytic RNA processing enzyme in Escherichia coli. We have sequenced a 3.2 kb EcoRI-BamHI fragment encoding the rne gene, and identified its reading frame. Upstream from the gene, there are appropriate consensus sequences for a putative promoter and a ribosome binding site. We have translated this gene using a T7 RNA polymerase/promoter system. We determined 25 amino acids from the N-terminal of the translated product and they are in full agreement with the DNA sequence. The translated product of the rne gene migrates in SDS containing polyacrylamide gels as a 110,000 Da polypeptide, but the open reading frame found in the sequenced DNA indicates a much smaller protein. The entity that migrates as a 110,000 Da contains RNA, which could account, at least partially, for the migration of the rne gene product in SDS containing polyacrylamide gels.
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PMID:Sequencing and expression of the rne gene of Escherichia coli. 201 93

The secondary structure of the 9S RNA precursor to ribosomal 5S RNA in Escherichia coli has been determined using chemical reagents and ribonucleases in combination with a reverse transcription procedure. The 9S RNA precursor was generated in vitro by T7 RNA polymerase, and the rrnB operon terminator, T1, was able to terminate the in vitro transcript. The secondary structure model exhibits three structural domains corresponding to a 5' region, a mature region and a terminator region. The mature domain is structurally identical to 5S RNA, and the ribosomal proteins L18 and L25 are able to bind to the precursor. The processing endoribonuclease RNase E cleaves between the structural domains. Moreover, an intramolecular refolding of the nascent transcript must take place if the current view of RNase III processing stems is correct.
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PMID:The 9S RNA precursor of Escherichia coli 5S RNA has three structural domains: implications for processing. 304 57

We have used either Escherichia coli or T7 RNA polymerase to transcribe in E. coli a series of lacZ genes that differ in the nature of their ribosome binding sites (RBS). Each T7 RNA polymerase transcript yields from 15- to 450-fold less beta-galactosidase than its E. coli polymerase counterpart, the ratio being larger when weaker RBS are used. The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E. coli RNA polymerase transcripts. T7 RNA polymerase transcribes the lacZ gene approximately 8-fold faster than the E. coli enzyme. We propose that this higher speed unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E. coli enzyme is used. This leads to degradation of the T7 transcript, unless the leading ribosome comes in time to shield the cleavage site: the weaker the RBS, the lower this probability and the more severe the inability of T7 RNA polymerase transcripts for beta-galactosidase synthesis.
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PMID:The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. 754 88

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.
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PMID:Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E. 759 53

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA. In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.
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PMID:Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation. 774 52

When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, the beta-galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the beta-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro. Aside from very slow mutants that were not sufficiently processive to transcribe the lacZ gene, the lower the polymerase speed, the higher the beta-galactosidase yield per transcript. In particular, a mutant which was 2.7-fold slower than the wild-type enzyme yielded 3.4- to 4.6-fold more beta-galactosidase per transcript. These differences in yield vanished in the presence of the rne-50 mutation and therefore reflect the unequal sensitivity of the transcripts to RNase E. We propose that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymerase, the longer the lag between the synthesis of this site(s) and its shielding by ribosomes, and the lower the transcript stability.
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PMID:Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed. 861 79

Previous work from this laboratory has shown that T7 RNA polymerase outpaces ribosomes in vivo, generating naked mRNA stretches which may be nuclease-sensitive. In particular, lacZ transcripts synthesised this way are highly unstable and yield little beta-galactosidase. We have argued that most of these transcripts are prematurely inactivated via an RNase E cleavage that occurs ahead of the leading ribosome, whereas a few escape this initial cleavage and are translated normally. Presumably, these rescued transcripts are later inactivated non-nucleolytically and subsequently scavenged by a process partially controlled by RNase E, as for the natural lacZ mRNA. In contrast, despite being synthesised by T7 RNA polymerase, T7 late transcripts are stable. The 5' regions of several of these transcripts, exemplified by the gene 10 mRNA, harbour hairpin structures which may act as barriers against RNase E action. To test whether these structures are indeed 5' stabilisers, we replaced the lacZ leader sequence by the corresponding region from T7 gene10. This replacement yielded a ca 2.9-fold increase in beta-galactosidase yield per transcript. This increase vanished in the presence of the rne-50 mutation which inactivates RNase E, and therefore it reflects a protection of the transcript against RNase E-dependent inactivation. Yet, the leader replacement did not stabilise the transcript chemically. We propose that this replacement inhibits the initial cleavage step but somehow facilitates the subsequent scavenging process.
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PMID:The lacZ mRNA can be stabilised by the T7 late mRNA leader in E coli. 891 30

Polyribonucleotide phosphorylase (PNPase) is one of the critical components of the E. coli RNA degradosome, which consists of both PNPase and endoribonuclease RNase E. The function of this complex is to control the rate of mRNA degradation. The PNPase possesses two enzymatic activities, namely 3'-5' processive exoribonuclease activity and 5'-3' RNA polymerase activity. In the present study, we used conventional chromatography to purify an E. coli protein that binds to a specific double-stranded DNA sequence. Microsequencing of the purified protein showed that this DNA-binding protein was PNPase. Our data further demonstrate that PNPase binds to DNA in a sequence-specific manner. These data suggest that PNPase may have previously unappreciated DNA-related functions in addition to its known role in mRNA degradation.
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PMID:Polyribonucleotide phosphorylase is a double-stranded DNA-binding protein. 950 33

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.
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PMID:The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. 1041 35


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