Retention of Core Catalytic Functions by a Conserved Minimal Ribonuclease E Peptide That Lacks the Domain Required for Tetramer Formation

Jonathan M Caruthers,Yanan Feng,David B Mckay,Stanley N Cohen

doi:10.1074/jbc.m602467200

Abstract

Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

Highlights

EXPERIMENTAL PROCEDURESPlasmids and Bacterial Strains—A plasmid encoding an E. coli N-Rne fragment consisting of amino acid residues 1– 498 followed by a hexahistidine affinity tag and a thrombin cleavage site in a pET16b vector (Novagen) has been described [17, 32]
Binding of other proteins that are assembled into a ribonucleolytic complex known as the degradosome (20 –23)
Earlier work has shown that the 498-residue amino-terminal segment of E. coli Ribonuclease E (RNase E) (i.e. N-Rne), which encompasses a fragment in which the amino acid sequence is highly conserved across all members of RNase E/G families, retains the structural features needed to enable the enzyme to cleave at specific RNA sites [17], confers a quasi-processive 3Ј to 5Ј scanning mode of enzymatic action [28, 42], and supports bacterial viability

Summary

EXPERIMENTAL PROCEDURES

Plasmids and Bacterial Strains—A plasmid encoding an E. coli N-Rne fragment consisting of amino acid residues 1– 498 followed by a hexahistidine affinity tag and a thrombin cleavage site in a pET16b vector (Novagen) has been described [17, 32]. To construct a plasmid expressing a fragment of H. influenzae RNase E, the coding sequence for residues Ϫ16 to 476 5 was PCR-amplified from genomic DNA of strain KW20 (American Type Culture Collection 51907) and cloned into the inteinbased expression vector pTYB4 (New England Biolabs) by ligation in a manner that added a Pro-Gly dipeptide at the carboxyl terminus of the final expressed protein product. Coding sequences for shorter fragments of the E. coli and H. influenzae RNase E enzymes were amplified from these plasmids and cloned by ligation into the inteinbased expression plasmid pTYB1 (New England Biolabs) in a manner that left no extraneous residues on the carboxyl termini of the protein products. Gene fragments were PCR-amplified using the plasmids described above as templates and were introduced into the NotI- and XbaI-cleaved Ampr pRNG3 parental plasmid [10] so that the expression of individual gene fragments is controlled by lacUV5 promoter. Fragments expressing E. coli RNase E residues 1 to 400 and 1 to 395, H. influenzae RNase E residues Ϫ16 to 476 and Ϫ16 to 399, E. coli RNase G residues 1 to 400, and H. influ-

The abbreviations used are

RESULTS

DISCUSSION