We celebrate the 60th anniversary of the Lac operon this year and what bouquet − et bien sûr un coup de champagne − would be more appropriate than to take a look at how post-transcriptional gene regulation complements and extends classical transcriptional gene regulation? I'm talking of small RNAs (sRNAs) and their role in post-transcriptional gene regulation (see here in STC for Matthias Gimpel's introduction to the world of small RNAs.) As an example suitable for the birthday celebration, I found one the other day that touches on the Lac operon.
Over the past two decades, sRNAs popped up as connecting links in regulatory chains of virtually all bacterial differentiation pathways such as virulence, biofilm formation, formation of flagella, sporulation, intercellular communication (quorum sensing), stress response, you name it. For completeness: the 'classical' chain links being transcription factors (including response regulators), sigma factors and nucleoid-associated proteins (NAPs). Evidence is accumulating that post-transcriptional gene regulation via sRNAs also affects central carbon metabolism in bacteria (see here for a review). The pinnacle of molecular tinkering with the cell's core metabolic functions, however, are the so-called "dual-function" sRNAs, "chimeric transcripts that serve as both: base-pairing riboregulators and mRNAs" as Kavyaa Venkat and coworkers from Kai Papenfort's lab at Jena University, Germany, say in their recent study of VcdRP, a sRNA of Vibrio cholerae.
Venkat et al. found VcdRP among a pool of candidate sRNAs in a screen for inhibitors of cholera toxin (CTX) expression in Vibrio cholerae. Closer examination of the VcdRP gene for structural elements (promoter, terminator) revealed an approximately 306 nucleotide-long primary transcript, VcdR, encompassing an internal open reading frame (orf) coding for a 29 residue peptide, VcdP (Figure 1). The primary transcript is processed in vivo by RNase E to yield several isoforms of the mature VcdR sRNA. In order to disentangle the possibly separate functions, they expressed VcdRP, VcdR, and VcdP (alone or as fusions to tags) from inducible multicopy plasmids in wild-type and ΔvcdPR hosts. They then measured, by RNA-Seq, alterations in transcript levels across the entire genome.
Indeed, pulse expression of VcdRP resulted in differential expression of 103 genes (84↑/19↓). VcdP modulated the expression of 49 genes (41↑/8↓), whereas VcdR led to a change in 8 genes (2↑/6↓). Most genes regulated by either VcdP (34/49) or VcdR (7/8) were also differentially expressed in response to VcdRP. However, regulation of 64 genes was specific to VcdRP, suggesting that simultaneous expression of VcdR and VcdP could have a synergistic effect on the cell. You can see from these numbers that VcdRP is certainly not a mono-specific regulator, an on/off switch, but a rather global one, more like a dimmer.
By functional annotation of the genes differentially expressed in response to VcdRP the authors detected several genes with potential functions in metabolic processes and specifically carbon metabolism. Functional assays with appropriate reporter fusions led them detect a common regulatory motif, a C quadruplet, in the 5'-untranslated region (5'-UTR) of the mRNAs of four sugar transporters, PtsG, NagE, TreB, and PtsHI, where base-pairing with VcdR would block the Shine-Dalgarno sequence (ribosome binding site (RBS)) and inhibit their translation. They could confirm this inhibition by experiments and conclude that indeed VcdR acts as post-transcriptional regulator of these genes (see Figure 1 for details).
To come full circle, or better: to close the regulatory loop: Venkat et al. found that CRP·cAMP binds to the VcdRP promoter (pvcdRP) in vitro and inhibits its expression in vivo. You may recall from the regulation of the Lac operon that CRP·cAMP activates expression of the lac genes − and those of other non-PTS sugars − under low glucose levels while lactose needs to be present to relieve their repression by the Lac repressor, LacI. Simultaneously, CRP·cAMP represses VcdRP expression thereby removing the translational inhibitor of several PTS/non-PTS sugar uptake mRNAs.
Almost en passant, you have here the explanation for the initial observation that overexpression of VcdRP inhibits CTX expression. It's long been known that the expression of a number of V. cholerae virulence genes, including the CTX genes, is triggered by the uptake of certain sugars. If uptake of these sugars is greatly reduced or impossible because the mRNAs of the corresponding sugar transporters are blocked for translation by binding of VcdRP, CTX expression levels remain low. Clearly, the inhibition of CTX expression by VcdRP is indirect (Figure 1).
VcdP, the small protein, interferes with carbon metabolism at different 'traffic junction'. It binds to citrate synthase, GltA, thus tuning the citric acid cycle at step 1. The exact molecular mechanism is not yet known but it seems that VcdP does not tinker with GltA levels but rather with its enzymatic activity (Figure 1). Kinetic measurements of both entry points, sugar uptake and TCA cycle, will have to be done to determine the degree of influence of VcdR and/or VcdP under specific growth conditions. So far, we just have a set list of the players.
Gimpel M, Brantl S. 2017. Dual-function small regulatory RNAs in bacteria. Mol Microbiol, 103(3), 387−397. PMID 27750368
Jacob F, Monod J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3, 318−356. PMID 13718526
Venkat K, Hoyos M, Haycocks JR, Cassidy L, Engelmann B, Rolle-Kampczyk U, von Bergen M, Tholey A, Grainger DC, Papenfort K. 2021. A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae. EMBO J, e108542. PMID 34612526
Some background noise...
A side note on "small RNAs": do not be misled by their name. With a length of 50−300 nt, sRNAs are only "small" in comparison to the "large" ribosomal RNAs (E. coli 16S: 1541 nt; 23S: 2,904 nt). As a rule of thumb, sRNAs are of the same size or even significantly larger with respect to molecular weight and molecular dimensions than the proteins they interact with. The "small" RNA of Vibrio mentioned here, VcdR, has a molecular weight of ~95 kDa while the repressor of pvcdRP, CRP protein, clocks in at ~24 kDa. The Bionumbers website (and book) gives another illuminating size comparison of myoglobin mRNA and the mature myoglobin protein (Figure 2). To avert a misunderstanding of "small", researchers suggested the general term 'non-coding RNA' (ncRNA) for all those transcripts that do not carry signals for translation, irrespective of their lengths. But this term is not very helpful because it is − like the term "prokaryote" − a definition ex negativo, and so it squeezes a whole zoo of RNA molecules with widely and wildly different functions under one umbrella (tRNA, rRNA, tmRNA, sRNA, gRNA (CRISPR!), microRNA (µRNA), siRNA, piRNA, snoRNA, snRNA, exRNA, scaRNA, ...).
Last but not least, the term 'ncRNA' does not help with VcdRP in particular, since the transcript is both a sRNA, VcdR, and a mRNA contained within, coding for VcdP. Therefore, it is best to pragmatically stick with the term "dual-function RNA" just as Venkat et al. (2021) did and focus on elucidating the various functions (Figure 1). Dual-function sRNAs are not exotic, they are found in Gram‑positives and Gram‑ negatives. But among the sRNAs they're a minority (see here for a review.)
A final note: VcdP, the 29 residue-long protein encoded within the VcdR sRNA, is really small (molecular weight ~3,200 kDa) and belongs to the size class of the smallest ribosomal proteins (L34 5.4 kDa, S17 9.4 kDa) but is still in another size class than the <10 residue-long oligopeptides used as signaling molecules by B. subtilis, for example.