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The quaternary structure of RNase G from Escherichia coli
Author(s) -
Briant Douglas J.,
Hankins Janet S.,
Cook Michael A.,
Mackie George A.
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03775.x
Subject(s) - rnase p , endoribonuclease , rnase ph , rnase mrp , biology , protein quaternary structure , biochemistry , serine , cysteine , protein subunit , escherichia coli , microbiology and biotechnology , enzyme , gene , rna
Summary 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.