Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation

The addition of poly(A)-rich sequences to endonuclease cleavage products of chloroplast mRNA has recently been suggested to target the polyadenylated RNA for rapid exonucleolytic degradation. This study analyzed whether the addition of a poly(A)-rich tail to RNA molecules is required for degradation by chloroplast exonuclease(s). In lyzed chloroplasts from spinach, addition of the polyadenylation inhibitor, cordycepin triphosphate (3′-dATP), inhibited the degradation of psbA and rbcL mRNAs. Furthermore, degradation intermediates generated by endonucleolytic cleavages accumulated. Similar results were obtained when yeast tRNA was added to the mRNA degradation system as a non-specific exoribonuclease inhibitor. Nevertheless, the stabilization mechanisms differ: while tRNA directly affects the exonuclease activity, 3′dATP has an indirect effect by inhibiting polyadenylation. The results indicate that the addition of poly(A)-rich sequences to endonucleolytic cleavage products of chloroplast mRNA is required to target these RNAs for rapid exonucleolytic degradation. Together with previous work, the data reported here support a model for mRNA degradation in the chloroplast in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the proximal cleavage products, targeting these RNAs for rapid exonucleolytic decay.     

Induction of macrolide-lincosamide-streptogramin B resistance requires ribosomes able to bind inducer

Summary Plasmids were constructed containing the regulatory regions and N-terminal portions of ermC and of ermD, fused in phase with the coding sequence of the Escherichia coli lacZ gene. ermC and ermD are erythromycin (Em) inducible macrolide-lincosamide-streptogramin B resistance elements derived from Staphylococcus aureus and Bacillus licheniformis, respectively. The fusion plasmids were introduced into B. subtilis and used to study ermC and ermD regulation. In both cases, -galactosidase synthesis could be induced by low levels of Em. Induction was prevented by introduction of ole-2, a chromosomal mutation which decreases ribosomal affinity for Em. Induction also did not occur in the presence of intact copies of ermC, suggesting that prior or concomitant methylation of 23S rRNA, a treatment known to decrease ribosomal affinity for Em, was capable of interfering with ermC and ermD induction. These experiments are consistent with the translational attenuation model of ermC regulation, and together with other evidence, suggest that ermD is regulated by a similar mechanism.     

The ribonucleases J1 and J2 are essential for growth and have independent roles in mRNA decay in Streptococcus pyogenes

The paralogous ribonucleases J1 and J2, recently identified in Bacillus subtilis, have both endoribonucleolytic and 5′‐to‐3′ exoribonucleolytic activities and participate in degradation and regulatory processing of mRNA. RNases J1 and J2 have partially overlapping target specificities, but only RNase J1 is essential for B. subtilis growth. Because mRNA decay is important in regulation of virulence factors of Streptococcus pyogenes (the group A streptococcus, GAS), we investigated the role of these newly described RNases in GAS. We found that conditional mutants for both RNases J1 and J2 require induction for growth, so we conclude that, unlike the case in B. subtilis, both of these RNases are essential for GAS growth, and therefore their functions are not redundant. We compared decay of representatives of the two classes of messages we had previously identified: Class I, which decay rapidly in exponential and stationary phase of growth (hasA and gyrA), and Class II, which are stable in stationary phase and exhibit a biphasic decay curve in exponential phase (sagA and sda). We report that RNases J1 and J2 affect the rate of decay of Class I messages and the length of the first phase in decay of Class II messages.     

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins

Summary The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH₂-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH₂-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis.Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH₂-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

Heterogeneity of the genes coding for 5 S RNA in three related strains of the genus Bacillus.

Summary Three related strains of the genus Bacillus, viz. B. licheniformis, B. subtilis and Bacillus Q were all found to contain a minor species of 5 S RNA in an amount indicating that it is transcribed from only one of the multiple 5 S RNA cistrons known to be present in these strains. The major and minor types of 5 S RNA from each of the three strains could be separated from each other by polyacrylamide gel electrophoresis in the presence of urea. The complete nucleotide sequences of the minor B. subtilis and Bacillus Q 5 S RNAs have been determined. Comparison of these sequences to the previously determined sequence of the minor 5 S RNA from B. licheniformis (Raué et al., 1976) showed the three minor types of 5 S RNA to be identical except for the residues at positions 79, 85 and 95. Moreover, seven out of the eight sequence differences between the major and the minor 5 S RNA are the same in all three strains. Thus, the single cistron coding for minor 5 S RNA has been strongly conserved in all three strains, which may indicate a biological significance for the minor 5 S RNA species.     

Rae1/YacP,a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis

The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo. Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo. Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro. Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo.     

A small RNA activates CFA synthase by isoform‐specific mRNA stabilization

Small RNAs use a diversity of well‐characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq‐associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation‐independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot‐exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E‐mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA‐controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.