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Ends of RNA Masking Transcription Termination Events in Prokaryotes

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Ends of RNA Masking Transcription Termination Events in Prokaryotes Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes Xun Wanga, Monford Paul Abishek Nb, Heung Jin Jeonb, Yonho Leeb, Jin Hea, Sankar Adhyac,1, and Heon M. Limb,1 aState Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China; bDepartment of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, 305-764 Daejeon, Republic of Korea; and cLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Sankar Adhya, December 10, 2018 (sent for review July 31, 2018; reviewed by Max E. Gottesman and Jeffrey W. Roberts) Two kinds of signal-dependent transcription termination and RNA Results release mechanisms have been established in prokaryotes in vitro Two Tandem Termination Signals at the End of gal. The end of the by: (i) binding of Rho to cytidine-rich nascent RNA [Rho-dependent gal operon is depicted in Fig. 1. The stop codon (UAA) of the termination (RDT)], and (ii) the formation of a hairpin structure in last ORF, galM, is successively followed by 6 bp, a dyad symmetry the nascent RNA, ending predominantly with uridine residues and three uridine residues in RNA, the latter two features be- [Rho-independent termination (RIT)]. As shown here, the two sig- coming a hairpin structure and three uridine residues in RNA nals act independently of each other and can be regulated (sup- which comprise a potential RIT signal. Downstream of this is – pressed) by translation transcription coupling in vivo. When not located a 78-bp C-rich region in the template strand which in suppressed, both RIT- and RDT-mediated transcription termination RNA becomes a potential RDT signal (11–13). do occur, but ribonucleolytic processing generates defined new 3′ ends in the terminated RNA molecules. The actual termination In Vitro and in Vivo Transcription in gal. The 3′ ends of specific events at the end of transcription units are masked by generation transcripts were identified and mapped both in vitro and in vivo ′ ′ of new processed 3 RNA ends; thus the in vivo 3 ends do not by using 3′ RACE. For in vitro transcription termination assays, ′ define termination sites. We predict generation of 3 ends of we used the plasmid pgal-gpmA as a DNA template containing mRNA by processing is a common phenomenon in prokaryotes the entire gal operon, as well as the next monocistronic operon as is the case in eukaryotes. gpmA with the same direction of transcription (SI Appendix, Figs. S1 and S2A). The 3′ RACE assays on the gal transcript generated transcription termination | RNA processing | gal operon | in vitro identified two major 3′ ends at positions 4396 and 4421 translation–transcription coupling (Fig. 2, lane 1) and two minor ends at 4313 and 4315. Since no obvious secondary structures or C richness are located upstream wo types of transcription termination mechanisms have been of the 3′ ends of the two major RNA, it is likely that these 3′ ends Tdocumented in prokaryotic organisms in vitro: (i) Rho- result from “elemental” transcription pausing that usually occurs dependent termination (RDT) facilitated by binding of Rho preceding actual transcription termination events (14–17). Se- protein to a cytidine-rich (C-rich) segment in the nascent RNA quence analysis of these sites also suggested that they contain a followed by dissociation of the RNA; and (ii) intrinsic or Rho- consensus core sequence for elemental pausing (18, 19). Of the independent termination (RIT) facilitated by formation of an minor RNA species without Rho, the one ending at 4315 (Fig. 2, RNA hairpin structure with five to seven uridine residues at the lane 1) is located seven nucleotides downstream from the foot of end that causes RNA release (1–7). We investigated the two the stem of the terminator hairpin (Fig. 1B). The 3′ end at 4315 types of termination events both in vivo and in vitro using the gal may be the result of the RIT signal which is functioning in- operon of Escherichia coli as a model system. efficiently (poor U richness). The transcription factors NusA and The gal operon is tetracistronic and is about 4 kb long. DNA NusG, respectively, stimulated and reduced the production of sequence shows that the end of gal contains both RIT and RDT signals in that order (Fig. 1). Northern analysis showed that the Significance full-length gal mRNA in vivo is about 4.3 kb long (termed mM1) and its 3′ end appears at the RIT (8). First, the RIT signal in gal Transcription termination by RNA polymerase in prokaryotes is has only three uridine residues and thus not expected to effi- well understood in contrast to similar mechanisms in higher ciently release RNA during termination. Second, the stop codon organisms. Despite the in vitro occurrence of two types of of the last ORF (galM) being only 6 bp away from the beginning demonstrable transcription termination events in prokaryotes of the RIT signal is too close to allow the required RNA hairpin at the end of transcription units, they are obscured in vivo in two ways: suppression of termination by traversing of the RNA structure formation because of translation–transcription cou- polymerase through the termination sites when coupled to pling (see below). Thus, it is not clear whether the mM1 RNA translation, or by further processing of the actual terminated really originates at RIT. There is also no evidence whether the RNA 3′ ends by RNases, as in eukaryotes. RDT signal is playing any role in gal. In fact, the actual func- tioning of both RIT and RDT signals in vivo has seldom been Author contributions: H.M.L. designed research; X.W., M.P.A.N., H.J.J., and Y.L. performed investigated in any system. We addressed this issue by in- research; X.W., M.P.A.N., H.J.J., Y.L., J.H., S.A., and H.M.L. analyzed data; and S.A. and H.M.L. wrote the paper. vestigating what role the two types of signals play in generating Reviewers: M.E.G., Columbia University; and J.W.R., Cornell University. the 3′ end of the mM1 mRNA in vivo. Since translation–tran- The authors declare no conflict of interest. scription coupling influences transcription termination (3, 9, 10), Published under the PNAS license. we also studied the role of the coupling in 3′ end formation. We 1To whom correspondence may be addressed. Email: [email protected] or hmlim@cnu. report the occurrence of transcription at the two types of signals, how ac.kr. – they are regulated by translation transcription coupling, and how the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. final 3′ end of the terminated RNA is actually generated by RNA 1073/pnas.1813181116/-/DCSupplemental. processing, thus, masking the actual termination processes. Published online February 19, 2019. 4440–4445 | PNAS | March 5, 2019 | vol. 116 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1813181116 Downloaded by guest on September 28, 2021 Fig. 1. (A) Schematic representation of the gal op- eron and the neighboring gpmA gene. Signals for the two tandem transcription terminations downstream of galM were shown. The RIT signal (the terminator hairpin) is presented as a cyan hairpin structure, and the RDT signal (C-rich region) is depicted as an orange line. Numbers indicate nucleotide position from the gal transcription initiation site, +1. E probe, which hybridizes to the first 500 nucleotides of galE,was used as the probe in Northern analyses throughout this study. (B) RNA sequences from the stop codon of galM to the start codon of gpmA. The terminator hairpin sequences are presented in cyan. The stem sequences are underlined. The C-rich region sequence is shown in orange. Numbers: 4285, the third nucleo- tide of the galM stop codon; 4313, the 3′ end of mM1 gal mRNA; 4393, the 3′ end of the C-rich region; and 4487, the first nucleotide of the gpmA start codon. the RNA with 3′ end at 4315, suggesting that the 4315 species is read-through transcription from the first RIT would stop at the GENETICS the result of RIT (Fig. 2A, lanes 3 and 4, respectively). NusA and second RIT while others would go further, to be stopped at the NusG have been previously established to stimulate and inhibit RDT. In the latter two cases, RNA would be processed back to RIT functions in general (17, 20–23). We believe that the other the stem and loop structure of the second RIT. The results minor band ending at 4313 (lane 1, Fig. 2A) mimicking in vivo gal showed that the RIT2 variant produced RNA with the 3′ end at mRNA mM1 and is generated by processing of the RNA ending the 4313 position as expected, but in reduced amounts, but also at 4315 by RNase present as contaminants of RNA polymerase made an RNA with the 3′ end at 4367 (Fig. 2B, lane 2). Re- (see below). In the presence of Rho, however, a major RNA duction of the 4313 band to about 50% of WT and the ap- appeared with a 3′ end at 4409 (Fig. 2A, lane 2). Since 4409 is pearance of about an equal amount of the 4367 RNA in RIT2 immediately downstream of the C-rich region (Fig. 1B), we suggest that 50% of transcription reaching the end of the gal conclude that Rho terminates transcription at 4409. operon terminates at the first RIT and the other 50% of tran- Total RNA extracted from wild-type (WT) E.
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