4 Discussion
4.2 Analysis of the rny suppressor mutants brings new insights into the regulation of the RNA
Taken into an account the very strong difference in the activity of the RNA polymerase variants in in vitro transcription assays, which was 200 fold for the RpoC-R88H variant as compared to the wild type polymerase and not even quantifiable for the RpoB-G1054C variant, we can ask ourselves whether such a huge decrease in RNA polymerase activity really occurs in vivo. Although even just 2-fold increase in the mRNA half-lives is apparently enough to get the RNA synthesis/degradation rate significantly out of equilibrium (Shahbabian et al., 2009), the more than 200-fold drop in transcription activity still seems to be too excessive. Although further experimental evidence will be needed to fully address this question, the decrease in transcription rates is likely milder in vivo. In the gram-negative model organism E. coli it is well established that the levels of RpoB and RpoC subunits of the RNA polymerase are subject to an auto-regulation on multiple levels (Dennis et al., 1985; Meek and Hayward, 1986). Whether the RNA polymerase
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subunits are subject to a similar auto-regulation also in B. subtlis has never been addressed.
Nevertheless, the presence of such auto-regulatory mechanism seems to be probable, not just as a rational explanation for the huge drop in in vitro transcriptional activity, but also judged from the increased protein quantity of the RNA polymerase RpoB subunit in the strains containing the RNA polymerase core mutations (both RpoB-G1054C and RpoC-R88H), which we have observed during Western-Blot experiments (data not shown).
In contrast to E. coli, where the rpoB and rpoC genes are part of a multicistronic operon together with ribosomal proteins, the B. subtilis rpoB and rpoC genes form just a bicistronic operon. This rpoBC operon is, however, preceded by a more than 200 bp long 5′ UTR which could have an influence on the rpoBC expression. Interestingly, a study published in the course of this thesis shown that RNase Y cleaves within this UTR to create an alternative 5′ end of the rpoBC transcript (DeLoughery et al., 2018), giving rise to a possibility that RNase Y is responsible for post-transcriptional regulation of rpoBC expression in B. subtilis. Such an observation also sparked the attractive speculation that the absence of this cleavage by RNase Y is the reason for the formation of suppressor mutation affecting the RNA polymerase in response to the rny deletion. That would falsify our previous conclusion about the pivotal function of RNase Y laying in the initiation of bulk mRNA degradation. However, such a possibility seems to be rather unlikely, since we did not observe any difference in the ß-galactosidase expression between the PrpoB-lacZ fusions containing or lacking the RNase Y cleavage site. Such a results suggests that the loss of the RNase Y cleavage site did not affect the expression of rpoBC genes.
Although our aforementioned model clearly show that the probability of assembly of the whole RNAP complex is lower when core subunits are duplicated (see Fig. 11), there is one factor which was for calculation simplicity left out during the model construction, but might play a role in the suppression mechanism, and this is the presence of alternative sigma factors. The housekeeping factor σA was the only sigma factor considered in the model, however, there are also 18 alternative sigma factors in B. subtilis. They are known to have lower affinity for the core than the housekeeping σA, which under normal circumstances contributes to the low expression of the genes under their control (Österberg et al., 2011). However, the alternative sigma factors may be favored in the situation with increased amount of uncomplete RNA polymerase complexes lacking some of the minor subunits. This was already shown on the example of rpoZ mutant in other organisms, which showed increased proportion of transcription dependent on alternative sigma factors (Geertz et al., 2011; Gunnelius et al., 2014). Hence, it is possible that the effect of the core duplication might not only lead to decrease of the overall transcription, but also increase the proportion of transcripts from promoters controlled by the alternative sigma factors.
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This would together account for the positive effect on the physiology of the rny suppressors, since alternative sigma factors are mainly involved in transcription of stress related genes which might help to combat the phenotypes caused by the rny deletion.
Whether this is really the case and alternative sigma factors play a role in the suppression has to be assessed in future. On one hand, one might expect that cells that need increased transcription of genes dependent on alternative sigma factors would simply upregulate expression of the sigma factor for instance by promotor up mutations. However, on the other hand, a simultaneous decrease of σA dependent transcription and increase in transcription from promoters controlled by multiple alternative sigma factors together might be most easily achieved by the duplication observed in our study, which is also supported by the finding that genomic amplifications are the easiest and most often occurring suppressing mutations in B. subtilis cells (Dormeyer et al., 2017; Reuß et al., 2019). One possible way to test the hypothesis about the alternative sigma factors involvement would be to introduce deletion of the rny gene into B. subtilis strain which was, on the other hand, proposed to have increased transcription activity from promoters dependent on the housekeeping sigma factor σA. That was shown for example for strains with rifampicin resistance variants of RpoB (Inaoka et al., 2004). If the hypothesis is correct, the rny deletion in such a background should lead to even more detrimental phenotype or obstacles in formation of suppressor mutations.
This thesis also brings strong support to the assumption that cold shock proteins, and especially CspD, actually are transcription factors. This can be deduced from the finding of cspD affecting mutations in the one class of suppressors next to the mutations in genes for the known transcription factor greA and the RNA polymerase subunit rpoE. This assumption is further supported by the evidence that CspB and CspD are localized around the nucleoid in transcription dependent manner (Weber et al., 2001). Despite its name, cspD is expressed stably at variety of conditions (Nicolas et al., 2012) and its role in transcription would be in agreement with the role of the homologous cold-shock proteins in the gram-negative model organism E. coli, for which an anti-termination activity was proposed (Bae et al., 2000). Whether CspD and other so-called cold shock proteins in B. subtilis also act as anti-terminator proteins or whether their role in transcription is different has to be subject of further investigations.
Another interesting finding this thesis brings about the cspD gene is the fact, that the suppressors with inactivated cspD gene seem to be genetically stable (see Fig. S3), in contrast to the progenitor rny mutant as well as the other suppressors evolved under different selection scenarios that do provide a growth benefit, but do not lead to complete genetic stabilization. It is not completely clear whether this genetic stabilization upon cspD inactivation is specific to the rny mutant background or whether CspD plays some general role in the cellular ability to evolve
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mutations. Preliminary results obtained on that topic in our laboratory suggest, that this is rather rny specific, since double deletion strain of cspD and cspB is forming suppressor extensively (Faßhauer and Stülke, unpublished). However, what is the exact link between RNase Y, CspD and the genome stabilization remains unclear.