Deciphering the RNA capping process in bacteria

RNA capping in eukaryotes has been studied since the 1970s, starting with the discovery of 5′ 7-methylguanylate caps in the Shatkin laboratory (1). That capping mechanism involves a pause during transcription elongation that allows the recruitment of specialized capping enzymes to modify the 5′ end of the nascent RNA. In bacteria, however, RNA capping was observed only within the past decade when derivatives of either coenzyme A (CoA) (2) or nicotinamide adenine dinucleotide (NAD) (3, 4) were reported at some RNA 5′ ends. The first bacterial capping mechanism was established convincingly in 2016 when it was observed that RNA polymerase (RNAP) could incorporate caps into RNA by using NAD and CoA as noncanonical initiating nucleotides (5). Last year, the Belasco laboratory at New York University demonstrated that a different type of cap, namely nucleoside tetraphosphate (Np₄), is found at the 5′ end of RNAs in Escherichia coli (6) under conditions where dinucleoside tetraphosphates (Np₄N) are increased. In PNAS, Luciano and Belasco (7) demonstrate the ability of E. coli RNAP to use Np₄N as the initiating nucleotide and its dependence on promoter sequence. In some cases, incorporation of an Np₄N is strongly favored over initiation with standard NTPs. They thereby establish that RNAP is the main Np₄ messenger RNA (mRNA) capping machinery used in E. coli and that the cap is incorporated during transcription initiation, in contrast with the postinitiation addition mechanism employed in eukaryotes.

Np₄A Alarmone Capping in Bacteria Has a Physiological Consequence

Multiple nucleotide-derived molecules function as stress signals, sometimes referred to as alarmones, in all three domains of life. The physiological roles of some of these alarmones have been well studied in bacteria. For example, guanosine tetraphosphate and pentaphosphate, abbreviated as (p)ppGpp, are signals that transform the transcriptome in response to changes in nutrient availability, a phenomenon known as the stringent response. In response to amino acid starvation in E. coli, (p)ppGpp dramatically inhibit transcription of hundreds of promoters and activate transcription of hundreds of other promoters by binding directly to RNAP (8). (p)ppGpp also bind to a large number of other enzymes, further altering the activities of the cell’s proteome (9). Likewise, cyclic di-GMP, another well-characterized alarmone, regulates a plethora of signaling pathways, thereby modulating virulence in numerous pathogens (10).

Although Np₄A alarmones were discovered decades ago as by-products of aminoacylation, and their occurrence is also widely conserved (11), their functions (if any) had remained elusive (12). The recent discovery of Np₄ caps at the 5′ end of mRNAs in E. coli (6) suggested that, rather than serving as regulators by binding to receptors, Np₄N signaling could alter regulatory output directly by modifying mRNAs. The Belasco group showed that disulfide stress increases levels of Np₄A via the inhibition of the Np₄A hydrolase ApaH, thereby affecting capping of multiple RNAs, a clear demonstration that an environmental change can alter RNA capping. Consistent with this observation, deletion of the apaH gene led to Np₄A accumulation (13) and increased capping (6).

Luciano and Belasco (7) show that many Np₄As can be used very efficiently as initiating nucleotides, both in vitro and in vivo. This capping mechanism is reminiscent of NAD and CoA capping of mRNAs (5), except that for all tested promoters incorporation of Ap₄A as the initiating nucleotide is much preferred over ATP (adenosine 5′-triphosphate) in in vitro transcription assays (7). Other Np₄As and even molecules with an alternative number of bridging phosphates (Np₅As and Np₃As, for example) were also preferred over ATP during transcription initiation at some promoters.

This is different from NAD capping and CoA capping for which the efficiency of incorporation was much lower than with ATP in every tested promoter (5). It is possible, of course, that optimal conditions for NAD and CoA 5′ capping have not yet been determined and that their incorporation efficiencies could reach those observed with Np₄As given the right conditions.

The Promoter Determinants of Np₄N RNA Capping and Regulation of Gene Expression

Promoter positions from −11 to +2 relative to the transcription start site are major determinants of transcription initiation efficiency. This region becomes single-stranded during open complex formation, affecting multiple steps in the mechanism (14, 15).

Luciano and Belasco (7) demonstrate that positions −3 through +1 on the template strand affect incorporation of Np₄As both in vitro and in vivo. The identity of the +1 nucleotide affects which Np₄A species can be incorporated, for example any of the four Np₄As when +1 is a T, and only Ap₄G, Ap₄C, or Ap₄U when +1 is a C, G, or A, respectively. The identity of the base at −1 seems to have the largest effect on incorporation of Np₄A; a template strand pyrimidine is highly favored for Np₄A incorporation.

Since the authors showed that Np4 caps affect rates of mRNA degradation (6), the effect of promoter sequence on capping provides a mechanism for differentially altering gene expression when Np4A levels are elevated. In addition, since all promoters tested so far initiate better with Np₄A than with ATP in vitro, and there are some promoters where the initiating NTP can be limiting for transcription (16), it is conceivable that transcription from those promoters might also be increased independent of the effect on mRNA stability, potentially providing an additional mechanism of regulation.

In summary, Np₄ capping provides an underappreciated mechanism for regulation of gene expression in bacteria. As its role is explored further, it seems likely that it will be found to play a role in responses to environmental and nutritional changes that have previously been unexplained.