Bacillus subtilis trp Leader RNA

Abstract

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AUrich region just upstream of the 3 transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3-to-5 exonuclease processing from the native 3 end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5 tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5 end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.Protective features of the 5Ј and 3Ј ends of prokaryotic mRNAs explain why these RNA molecules are not degraded immediately by the multiple ribonucleases that are present in a prokaryotic cell. The presence of a nucleoside triphosphate at the 5Ј end renders this end a poor substrate for 5Ј-to-3Ј exonucleolytic decay (1-3) or 5Ј end-dependent endonucleolytic activity (4 -6). The strong stem loop structure found at the 3Ј end of many prokaryotic mRNAs, which is the transcription terminator structure, is resistant to 3Ј-to-5Ј exonucleolytic decay (7,8). A general model for the initiation of mRNA decay in prokaryotes, which is based on numerous studies in Escherichia coli, is as follows: initiation of decay occurs by an endonucleolytic cleavage in the body of the message. Such cleavage generates an upstream fragment with an unprotected 3Ј end, which is a good substrate for 3Ј-to-5Ј exonucleases, and a downstream fragment with a monophosphate nucleoside 5Ј end, which is a good substrate for additional endonuclease cleavages (9 -11). The downstream fragment could also be a good substrate for a 5Ј-to-3Ј exonuclease activity; however, such an activity is not known to exist in E. coli.The major decay-initiating endonuclease in E. coli is believed to be RNase E, a 5Ј end-dependent endonuclease. That is, RNase E endonuclease activity is usually contingent on prior binding to the 5Ј terminus, after which the enzyme tracks or loops to its target cleavage site (5, 12). RNase E vastly prefers an RNA substrate with a monophosphate 5Ј end (4, 5, 13)...

5′ End-independent RNase J1 Endonuclease Cleavage of Bacillus subtilis Model RNA

“…2A shows the half-lives for these RNAs in the RNase J1 mutant strain. We observed previously that lowering cellular RNase J1 concentration resulted in a Ͼ2-fold increase in half-life (to 5.4 min) for wild-type trp leader RNA (8). Here, we found this to be the case also for 5Ј-SSS RNA (5.6 min), again suggesting that placement of the strongly structured stem-loop at the 5Ј end had no effect on RNA decay.…”

“…Plasmids-trp leader RNA mutants were carried on derivatives of plasmid pGD5 (6), which contains the wild-type trp leader sequence and the mtrB gene encoding TRAP, and were constructed as described (8). The selective marker on the initial set of plasmids was tetracycline resistance.…”

“…The possible involvement of RNase J1 5Ј-exonuclease activity in trp leader RNA decay was suggested by the fact that addition of an A 7 sequence to the 5Ј end of trp leader RNA reduced its half-life from 2.5 to 0.9 min (8). Similarly, addition of A 7 to the 5Ј end of NotI RNA reduced its half-life from 10.3 to 3.2 min.…”

“…5, B and C), it is possible that the rate of such cleavage may be lower for trp:rpsO-TT. It is also possible that there is a stabilizing effect of the stronger 3Ј-TT structure of rpsO, which could provide better protection against a minor 3Ј exonucleolytic decay pathway from the 3Ј end (8). In the RNase J1 mutant strain, trp:rpsO-TT RNA half-life increased 2.3-fold to 8.2 min (Table 1), matching almost exactly the 2.2-fold increase in halflife observed for trp leader RNA in the RNase J1 mutant strain ( Fig.…”

“…Previous studies (5)(6)(7)(8) on trp leader RNA have led to the following model for its turnover: initiation of decay occurs by RNase J1 cleavage at around nt 101, which is located in an AUrich sequence AUUAUGUUUAUU (nt 101 is underlined). Endonucleolytic cleavage at nt 101 leaves an upstream fragment with a susceptible 3Ј end that is attacked primarily by the 3Ј-to-5Ј-exonuclease, polynucleotide phosphorylase (PNPase).…”