No abstract

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5'-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping, and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

RNase E was originally discovered as an RNA-processing enzyme (1). Later it was found that its gene, rne, and ams (altered messenger stability) are identical (7,23,31), suggesting a key role for RNase E in bacterial mRNA processing and decay. Subsequent studies revealed that RNase E controls the stability of many (8, 9), if not most, mRNAs in Escherichia coli (3), as well as being involved in the processing of 5S and 16S rRNAs (1,20,32), tRNA precursors (19) and degradation of small regulatory RNAs (21). Moreover, RNase E has the ability to affect protein degradation through its role in maturation of the 3' end of tmRNA (SsrA) (22).Purification studies have revealed that in E. coli RNase E forms a large multienzyme complex referred to as 'degradosome ' (5, 24, 26). In addition to RNase E, the other major components of this complex are polynucleotide phosphorylase (pnpase), enolase and RhlB RNA helicase. Previous work has shown that the catalytic domain of the enzyme is conserved within the RNase E/G family; however, little or no similarity has been found regarding RNase E domains involved in the interactions of these proteins with other components of the degradosome (16). Although the degradation and processing of RNA in E. coli is relatively well understood, very little is known about the mechanisms involved in the RNA metabolism in other species (11,12), including mycobacteria. Previous work has revealed that mycobacteria are very successful pathogens (28), which are able to survive under adverse conditions (long non-replicating persistence, and intracellular survival). Given the central role of RNase E in RNA processing and decay in E. coli, it is conceivable that the RNase E-dependent regulation of RNA stability in mycobacteria might also be a very important mechanism, adjusting the cellular metabolism to environmental changes. To learn more about RNase E proteins that function in different mycobacterial species, we cloned, overexpressed and purified putative mycobacterial RNase E and identified proteins associated with this polypeptide.A 2.8 kb fragment containing the rne gene (Rv2444c) was amplified by PCR, using the oligonucleotide primers myc1 5'-CTG TGC ATA TGA TAG ACG GTG CCC-3' and myc2 5'-CGG AGA TCT GGT CAG TCT AGG CGG-3', with Mycobacterium tuberculosis H37Rv DNA as template. Oligonucleotides were designed by using the complete sequence of the M. tuberculosis H37Rv genome (10, 13). PCR amplification conditions were as recommended by the manufac- Abstract: RNase E and its complex with other proteins ('degradosome') play an important role in RNA processing and decay in Escherichia coli and in many other bacteria. To identify the proteins which can potentially interact with this enzyme in mycobacteria, Mycobacterium tuberculosis H37Rv RNase E was cloned and expressed as a 6HisFLAG-tagged fusion protein. Analysis of the mycobacterial RNase E overexpressed and purified from M. bovis BCG revealed the presence of GroEL and two other copurified proteins, products of the Mb1721 (inorganic polyphosphate/ATP-...