Em-induced ribosome stalling on the ermC
leader peptide CDS has been the subject of many previous studies, as it is a key step in this classic example of gene regulation by translational attenuation (Dubnau, 1984
, Weisblum, 1985
). Stalling of an Em-bound ribosome also serves as a model for studies of the inhibitory action of Em (Tenson et al., 2003
). Our results with tRNA probing demonstrate that I9 is the final amino acid incorporated in the peptide chain before Em-dependent ribosome stalling. These experiments did not, however, distinguish whether the I9 codon is in the A site (i.e., pre-translocation), as proposed by Mayford and Weisblum (1989)
, or the P site (i.e., post-translocation). While this manuscript was in revision, a report by Mankin and colleagues appeared in which an in vitro
system containing E. coli
ribosomes was used to show that Em-dependent stalling occurs with codon 9 in the P site and codon 10 in the A site (Vazquez-Laslop et al., 2008
). In addition, the data in this paper suggested that the accumulated peptidyl-tRNA was not a drop-off product. Assuming the results of the in vitro E. coli
system are applicable to the in vivo
situation in B. subtilis
, we can conclude that Em-dependent stalling leaves a ribosome with codon I9 in the P site and codon S10 in the A site. Thus, Em-induced ΔermC
mRNA processing, which occurs between codons 4 and 5, is not an example of A-site cleavage. It remains to be determined whether A-site cleavage occurs in B. subtilis
The question of which ribonuclease activity is responsible for Em-dependent processing was addressed in these studies. Experiments showed that a reduction in cellular RNase J1 content resulted in a significant decrease in the level of ΔermC
processing (). The fact that secondary structure located at the 5′ terminus interfered with Em-induced ΔermC
processing () indicated a 5′-end-dependent ribonuclease activity, of which RNase J1 is the only known example in B. subtilis
(de la Sierra-Gallay et al., 2008
, Even et al., 2005
, Mathy et al., 2007
). Note that the previous studies on the effect of 5′ secondary structure on ΔermC
mRNA half-life (Sharp & Bechhofer, 2005
) were performed in the absence of Em. Thus, both the turnover of ΔermC
mRNA and the processing upon ribosome stalling are inhibited by 5′-terminal structure. The simplest interpretation of these results is that RNase J1 is directly responsible for initiation of ΔermC
mRNA decay and for cleavage at the RSS. The effect of 5′ structure on RNase J1 activity needs to be addressed systematically.
Experiments shown in suggest that RNase J1 processing of ΔermC
mRNA is by endonucleolytic cleavage between codons 4 and 5, and not exonucleolytic decay from the 5′ end up to the stalled ribosome. As such, the RNase J1 recognition site may consist of specific ΔermC
nucleotides around codons 4-5, and RNase J1 may cleave at this site constitutively. In the absence of the stalled ribosome, such cleavage would initiate decay of the downstream fragment, likely by RNase J1 switching to 5′-to-3′ exonuclease mode and rapidly degrading the downstream 209-nt RNA, in accordance with a model that we and others have proposed for the role of RNase J1 in B. subtilis
RNA turnover (de la Sierra-Gallay et al., 2008
, Deikus et al., 2008
). In the presence of the stalled ribosome, the 5′ end of the 209-nt RNA would be protected from attack by RNase J1, and this RNA fragment would accumulate.
Protection of the 5′ end of the 209-nt RNA by the stalled ribosome is consistent with structural studies of mRNA:ribosome interactions. Using Thermus thermophilus
ribosomes, Noller and colleagues have determined that the section of mRNA that is wrapped around the ribosome “neck,” and interacts most closely with the ribosome, stretches from 6 nts upstream to 3 nts downstream of the A site (Yusupova et al., 2001
). In our case, cleavage between codons 4 and 5 would generate a 5′ end that is 15 nucleotides upstream of the A site or 9 nts upstream of the mRNA sequence that is most closely associated with the ribosome. Exonuclease progression is often blocked several nts away from a bound protein or an RNA secondary structure, so our results are consistent with the stalled ribosome constituting a block to further RNase J1-mediated 5′- to-3′ exonucleolytic degradation after an initial endonuclease cleavage. The binding cleft in RNase J1, in which the 5′ end of an RNA is thought to be situated for catalysis (de la Sierra-Gallay et al., 2008
), may not be able to accommodate an RNA 5′ end that is surrounded by ribosomal structural components.
An alternative explanation for ΔermC mRNA processing is that RNase J1 does not catalyze endonuclease cleavage directly but is required to activate some other ribonuclease activity, perhaps one that is ribosome-associated. Our in vitro results (), which indicate recognition of the cleavage site contained in codons 3-6, are more consistent with direct cleavage by RNase J1.
A noteworthy aspect of this study is the finding that ribosome stalling occurred in the ΔermC
construct that had a FLAG tag inserted immediately after the initiation codon. This insertion moved the RSS further away from the start of translation, such that Em-induced stalling was occurring with codon 17 in the P site (). This result is quite surprising in view of the work of others on the mechanism of Em inhibition of translation and ribosome stalling (Tenson et al., 2003
, Vazquez-Laslop et al., 2008
). Their results indicated that the RSS must be located at a particular distance from the N-terminus, optimally ending at codon 9. Translation of an mRNA, in the presence of Em, that had an RSS located further than codon 9 would likely result in peptidyl-tRNA drop-off before the stall site was reached. Our results, on the other hand, suggest that an Em-bound ribosome can synthesize a nascent peptide twice that size (17 amino-acids) before it reaches the stalling site. Further studies will be required to understand the structural basis of this finding, including the determination whether the FLAG-tag sequence is unique in allowing stalling further downstream.