Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes

Abstract

Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (ii) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation–transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3′ ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3′ RNA ends; thus the in vivo 3′ ends do not define termination sites. We predict generation of 3′ ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic gal mRNA

“…These results thus suggest that the 5 portion of the galETKM mRNA after the RNase E cleavages is removed immediately, possibly by 3 →5 exoribonucleases, as has been demonstrated in many previous studies (Mott et al, 1985;Belasco et al, 1986;Hui et al, 2014;Wang et al, 2019).…”

“…In the RIT o 2 mutant, two bases from the bottom of the stem of the terminator hairpin are substituted to their complementary bases ( Figure 1C). In the RIT o 2 mutant, the function of the terminator hairpin in the blockage of 3 → 5 exoribonuclease was impaired (Wang et al, 2019). When RNA from the RIT o 2 mutant was analyzed using the M-probe, we confirmed that the mutations caused the shortening of the galETKM mRNA by about 100 nucleotides and also decreased the galETKM amount to about 40% of the wild type (WT; lane 2 in Figure 1D; Wang et al, 2019).…”

“…In the RIT o 2 mutant, the function of the terminator hairpin in the blockage of 3 → 5 exoribonuclease was impaired (Wang et al, 2019). When RNA from the RIT o 2 mutant was analyzed using the M-probe, we confirmed that the mutations caused the shortening of the galETKM mRNA by about 100 nucleotides and also decreased the galETKM amount to about 40% of the wild type (WT; lane 2 in Figure 1D; Wang et al, 2019). The northern blot also showed that galTKM, galKM, and galM1 also got similarly shorter than their corresponding WT counterparts (lane 2, Figure 1D), and the amount of these mRNA species decreased to less than 10% of their amounts in the WT.…”

“…Also shown is the terminator hairpin. The cytosine residue at 4,313 is the last residue of the full-length mRNA, galETKM (Wang et al, 2019).…”

“…In either case, RNase E cleavage results in the generation of the 5 portion of mRNA with a new 3 -OH end and the 3 portion of mRNA with a new 5 end. Generally, the 5 portion mRNA is degraded immediately by 3 → 5 exonuclease digestion (Mott et al, 1985;Belasco et al, 1986;Hui et al, 2014;Wang et al, 2019). The 3 portion mRNA could be subjected to decay by further rounds of cleavage by RNase E by either of the pathways (Joyce and Dreyfus, 1998;Spickler et al, 2001;Hui et al, 2014).…”