The quaternary structure of RNase G from <i> Escherichia coli</i>

Briant, Douglas J.; Hankins, Janet S.; Cook, Michael A.; Mackie, George A.

doi:10.1046/j.1365-2958.2003.03775.x

Cited by 28 publications

(35 citation statements)

References 40 publications

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“…Assuming a crosslinking efficiency of 63% for multimeric RNase G under these reaction conditions, the data suggest that, unlike N-RNase E, this protein exists almost entirely as a dimer or higher-order multimer at the concentrations tested. This finding is consistent with recent sedimentation velocity studies (16) and with the lack of a significant effect of enzyme concentration on the marked 5Ј-end dependence of RNase G within this concentration range (Fig. 4B).…”

Section: Resultssupporting

confidence: 82%

“…Of relevance to this hypothesis are recent studies showing that RNase G exists primarily as a homodimer at moderate protein concentrations (16) and that N-RNase E exists primarily as a homotetramer at very high protein concentrations (17). That N-RNase E at lower concentrations can exist as a mixture of protein forms in different multimeric states was indicated by the results of gel electrophoresis under nondenaturing conditions, which revealed both monomeric and dimeric forms of the protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

Jiang

Belasco

2004

Proc. Natl. Acad. Sci. U.S.A.

110

112

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RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5 end. To investigate the mechanism by which 5 -terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5 monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5 monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5 phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5 -end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.M essenger RNAs are labile molecules whose longevity directly influences the synthesis rates of the proteins they encode. Despite the importance of mRNA degradation for gene expression, little is understood about the molecular mechanisms that govern differences in mRNA stability, even in so well studied an organism as Escherichia coli. Elucidating these mechanisms is crucial for explaining how RNA sequence and structure control mRNA lifetimes.The degradation of most E. coli mRNAs is thought to begin with internal cleavage by the endonuclease RNase E (1), although in some cases decay begins instead with cleavage by RNase III or RNase G (an RNase E homolog) (2, 3). The resulting mRNA fragments are then degraded to mononucleotides by a combination of further endonucleolytic cleavage and 3Ј exonucleolytic digestion.RNase E is a 1,061-aa protein comprising functionally distinct domains. The catalytically active amino-terminal half of the protein (N-RNase E: residues 1-498) is alone sufficient for the enzyme's ribonuclease activity, whereas the carboxyl half of the protein (residues 499-1061) contains both an arginine-rich region and a carboxy-terminal domain that serves as a scaffold for the assembly of a multiprotein complex known as the RNA degradosome (4, 5). By contrast, RNase G, which comprises 489 amino acid residues, is similar in sequence to the amino half of RNase E but lacks an arginine-rich region and a scaffold domain (6, 7).In view of the homology of RNase G to the catalytic domain of RNase E, it is not surprising that they have a similar cleavage site specificity, with both preferring to cut RNA within singlestranded regions that are AU-rich (8, 9). Another important property shared by these two en...

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

Jiang

Belasco

2004

Proc. Natl. Acad. Sci. U.S.A.

110

112

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“…In addition, as predicted from the experiments in B. subtilis (Pellegrini et al, 2003), the enzyme does not process a B. subtilis tRNA precursor sibly RNase Z and RNase LS, the most likely explanation for their apparent supporting role in mRNA decay in single mutants (Table 1, Ow et al, 2003;Otsuka and Yonesaki, 2005) arises from the relatively low abundance of these proteins. Clearly this is probably the case with RNase G (Briant et al, 2003), where overproduction of the protein was shown to decrease the half-life of the rpsT mRNA in an rne-1 mutant (Ow et al, 2003). Alternatively, initial RNase E cleavages may generate substrates that are more efficiently cleaved by RNase Z, RNase G and RNase LS.…”

Section: Discussionmentioning

confidence: 99%

RNase Z in Escherichia coli plays a significant role in mRNA decay

Perwez

Kushner²

2006

Molecular Microbiology

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SummaryGenetic and biochemical analysis of RNase Z in eukaryotes, such as Arabadopsis thaliana , and prokaryotes like Bacillus subtilis have demonstrated that this endoribonuclease is essential for the maturation of tRNA precursors that do not contain a chromosomally encoded CCA determinant. As all Escherichia coli tRNA transcripts have chromosomally encoded CCA determinants, the function of its putative RNase Z homologue, the product of the elaC gene, is not clear. Here we demonstrate that the E. coli ElaC protein (RNase Z) endonucleolytically processes B. subtilis tRNA precursors lacking a CCA determinant both in vivo and in vitro . More importantly, E. coli RNase Z plays a significant role in mRNA decay, a previously unidentified activity for the enzyme. The purified RNase Z protein cleaves the rpsT mRNA at locations distinct from those obtained with RNase E. As expected, under physiological conditions E. coli and B. subtilis tRNA precursors containing a CCA determinant are not substrates. These results suggest a potentially important new role for the RNase Z family of proteins in RNA metabolism, particularly in organisms lacking RNase E.

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“…3). Furthermore, not withstanding previous reports that six extra amino acids at the amino terminus of RNase G (a situation that does not occur in vivo) (Briant et al 2003) led to complementation of RNase E mutants (Lee et al 2002;Deana and Belasco 2004), this modified protein did not effectively complement the rne-1 allele at 44°C in the MG1693 genetic background (Fig. 3).…”

Section: Discussionmentioning

confidence: 72%

“…The chromosomal rng sequence is shown at the top. The native translation start, identified by sequencing of the protein purified from E. coli (Briant et al 2003) is shown as +1. The translation start site employed by Lee et al (2002) and Deana and Belasco (2004) is indicated as À18.…”

Section: Resultsmentioning

confidence: 99%

Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

Chung

Min²,

Wang³

et al. 2010

RNA

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The endoribonuclease RNase E of Escherichia coli is an essential enzyme that plays a major role in all aspects of RNA metabolism. In contrast, its paralog, RNase G, seems to have more limited functions. It is involved in the maturation of the 59 terminus of 16S rRNA, the processing of a few tRNAs, and the initiation of decay of a limited number of mRNAs but is not required for cell viability and cannot substitute for RNase E under normal physiological conditions. Here we show that neither the native nor N-terminal extended form of RNase G can restore the growth defect associated with either the rne-1 or rneD1018 alleles even when expressed at very high protein levels. In contrast, two distinct spontaneously derived single amino acid substitutions within the predicted RNase H domain of RNase G, generating the rng-219 and rng-248 alleles, result in complementation of the growth defect associated with various RNase E mutants, suggesting that this region of the two proteins may help distinguish their in vivo biological activities. Analysis of rneD1018/rng-219 and rneD1018/rng-248 double mutants has provided interesting insights into the distinct roles of RNase E and RNase G in mRNA decay and tRNA processing.

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The quaternary structure of RNase G from Escherichia coli

Cited by 28 publications

References 40 publications

Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

RNase Z in Escherichia coli plays a significant role in mRNA decay

Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

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