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Cited by 56RNA and protein synthesis mechanismsBacterial Genetics and BiotechnologyRNA Research and Splicing

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Substrate Binding and Active Site Residues in RNases E and G

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Garrey, Stephen M., Blech, Michaela, Riffell, Jenna L., Hankins, Janet S., Stickney, Leigh M., Diver, Melinda, Hsu, Ying-Han Roger, Kunanithy, Vitharani, Mackie, George A.·2009·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.063263PMC PMC2797255
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Abstract

The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5'-sensor, the S1 domain, and the Mg(+2)/Mn(+2) binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k(cat) decrease approximately 40- to 100-fold) and RNA binding consistent with a key role for the 5'-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.

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Metadata

Authors
Garrey, S. M., Blech, M., Riffell, J. L., Hankins, J. S., Stickney, L. M., Diver, M., Hsu, Y.-H. R., Kunanithy, V., & Mackie, G. A.
Affiliations
University of British Columbia (CA)
Corresponding author
Mackie, George A.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2009-11
Volume
284
Issue
46
Pages
31843-31850
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109.063263
PMC
PMC2797255
OpenAlex
W2042046941
Cited by
56
References
42
Citations by year
2026: 1 · 2025: 1 · 2024: 3 · 2023: 2 · 2021: 7 · 2020: 4 · 2019: 5 · 2018: 2 · 2017: 2 · 2015: 5 · 2014: 5 · 2013: 4 · 2012: 4
FWCI
1.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Canadian Institutes of Health Research (MOP-5396)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
RNase E, RNase G, endoribonuclease, RNA binding sites, S1 domain, active site residues, site-directed mutagenesis, catalytic efficiency, RNA metabolism, Escherichia coli
MeSH terms
Binding Sites, Endoribonucleases, Escherichia coli, Models, Molecular, Mutation, Phosphates, Protein Conformation, RNA, Bacterial, RNA, Ribosomal, 5S, Structure-Activity Relationship, Substrate Specificity, Blotting, Northern, Catalytic Domain, Escherichia coli Proteins

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Garrey, S. M., Blech, M., Riffell, J. L., Hankins, J. S., Stickney, L. M., Diver, M., Hsu, Y.-H. R., Kunanithy, V., & Mackie, G. A. (2009). Substrate Binding and Active Site Residues in RNases E and G. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.m109.063263

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