Mycobacterial RNase E‐Associated Proteins

Abstract

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-...

From conformational chaos to robust regulation: the structure and function of the multi-enzyme RNA degradosome

“…Indicated are the delta, beta, alpha and gamma branches of proteobacteria. For the species marked with an asterisk, association of RNase E with one or more degradosome components was confirmed experimentally (Ait- Bara & Carpousis, 2010 ;Erce et al 2009Erce et al , 2010Hardwick et al 2010 ;Jäger et al 2001 ;Kovacs et al 2005 ;Purusharth et al 2005 ;Yang et al 2008). Within gammaproteobacteria, Enterobacteriales, Pasteuralles and Vibrionales are predicted to have an E. coli type of degradosome, while Aeromonadales and Alteromonadales degradosomes lack enolase (Ait- Bara & Carpousis, 2010).…”

Composition and conservation of the mRNA-degrading machinery in bacteria

“…These studies revealed that, similar to their E. coli counterpart, RNase E/G homologues can interact with PNPase in Streptomyces [55] and are able to co-purify with GroEL and metabolic enzymes in Mycobacteria [56]. The specific role of these polypeptides in RNA metabolism and the degree, to which their interaction with RNase E/G is conserved in Actinobacteria, remains to be established.…”

TheCorynebacterium glutamicumNCgl2281 Gene Encoding an RNase E/G Family Endoribonuclease Can Complement theEscherichia coli rng::catMutation but Not therne-1Mutation

“…36) Another study has suggested that the mycobacterial RNase E/G homolog forms a degradosome-like complex. 37) These two studies suggest that the mycobacterial RNase E/G is functionally related to E. coli RNase E. S. coelicolor also has one RNase E/G homolog, which complemented both the E. coli rne-1 and the rng::cat mutation. 29,30) Bacillus subtilis has no RNase E/G homolog.…”