The quaternary structure of RNase G from Escherichia coli.
RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.
Briant
Douglas J
DJ
Department of Biochemistry and Molecular Biology, D H Copp Building, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
Hankins
Janet S
JS
Cook
Michael A
MA
Mackie
George A
GA
D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D020836:Protein Structure, Quaternary; D012329:RNA, Bacterial; D024363:Transcription Initiation Site; D012407:rRNA Operon
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D004722:Endoribonucleases; C081905:rng protein, E coli; D003545:Cysteine