Rho-independent transcription terminators inhibit RNase P processing of the secG leuU and metT tRNA polycistronic transcripts in Escherichia coli

Abstract

The widely accepted model for the processing of tRNAs in Escherichia coli involves essential initial cleavages by RNase E within polycistronic transcripts to generate pre-tRNAs that subsequently become substrates for RNase P. However, recently we identified two polycistronic tRNA transcripts whose endonucleolytic processing was solely dependent on RNase P. Here we show that the processing of the secG leuU and metT leuW glnU glnW metU glnV glnX polycistronic transcripts takes place through a different type of maturation pathway. Specifically, RNase P separates the tRNA units within each operon following the endonucleolytic removal of the distal Rho-independent transcription terminator, primarily by RNase E. Failure to remove the Rho-independent transcription terminator inhibits RNase P processing of both transcripts leading to a decrease in mature tRNA levels and dramatically increased levels of full-length transcripts in an RNase E deletion strain. Furthermore, we show for the first time that RNase G also removes the Rho-independent transcription terminator associated with the secG leuU operon. Our data also demonstrate that the Rne-1 protein retains significant activity on tRNA substrates at the non-permissive temperature. Taken together it is clear that there are multiple pathways involved in the maturation of tRNAs in E. coli.

Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

“…and cspA mRNAs also indicate that the Rne-1 protein retains a significant amount of residual activity at the nonpermissive temperature, a conclusion also supported by the analysis of tRNA processing by Mohanty and Kushner (2008). In contrast, the half-lives of the rpsT (rpsT P1 and rpsT P2 ) and cspE transcripts did not change significantly among the rneD1018/rne-1, rneD1018/rng-219, and rneD1018/rng-248 strains under any condition tested (Table 3), supporting previous results that other ribonucleases such as RNase G and RNase Z can initiate the decay of these transcripts (Ow et al 2003;Perwez and Kushner 2006).…”

“…In addition, rne-1 strains retain a significant amount of residual RNase E activity at 44 o C (Mohanty and Kushner 2008). Thus, the rneD1018/rng-219 and rneD1018/ rng-248 strains provided a means to test if the initiation of endonucleolytic decay of specific mRNAs was absolutely dependent on the presence of RNase E. For our analysis, we determined the half-lives of four mRNAs previously demonstrated to decay in an RNase E-dependent fashion (rpsO, rpsT, cspA, and cspE) (Mackie 1991;Hajnsdorf et al 1994;Perwez and Kushner 2006;Hankins et al 2007).…”

“…In fact, the essential function of RNase E was suggested to involve its role in the initiation of tRNA maturation (Li and Deutscher 2002;Ow and Kushner 2002;Perwez et al 2008), although Deana and Belasco (2004) have challenged this hypothesis. However, all the published experiments examining the role of RNase E in tRNA maturation have been carried out using strains that either contained some RNase E activity (rneD610, rneD645, or rne under the control of a lac promoter) or retained residual activity at the nonpermissive temperature (rne-1 and rne-3071) (Mohanty and Kushner 2008 Pro were chosen because their maturation is highly dependent on RNase E (Li and Deutscher 2002;Ow and Kushner 2002). In contrast, tRNA…”

The Presence of a C−1/G+73 Pair in a tRNA Precursor Influences Processing and Expression In Vivo

“…12 It has been suggested that Rho-independent transcription terminators inhibit the RNase P processing step. 33 However, based on the in vivo data presented here (Fig. 4), where we compared the efficiency of the processing of constructs with and without the Rho-independent terminator, its presence does not appear to have any significant effect on the processing of the tRNA precursor.…”

Corynebacterium glutamicum RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5′ maturation of 5S rRNA

“…coli RNase E is involved in the initial processing of many tRNA transcripts, providing substrates for final maturation by RNase P and 3 0 exoribonucleases (Li and Deutscher 2002;Mohanty and Kushner 2007;Mohanty and Kushner 2008;Ow and Kushner 2002). E. coli RNase E is also required for the maturation of tmRNA (Lin-Chao et al 1999).…”