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Title: A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5′ RNA-seq}
Article:
Abstract:
Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, “primer-dependent initiation, ”( PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5′ ends( 5′ RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T−1A+1 or G−1G+1( where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.
Introduction:
Transcription in all cells is carried out by multi-subunit RNA polymerases( RNAPs) that are conserved in sequence, structure, and function from bacteria to humans. The first step in transcription, initiation, consists of a number of discrete steps that culminate in the RNAP-mediated catalysis of the first phosphodiester bond formed within the nascent RNA[1, 2]. The first phosphodiester bond within the nascent RNA can be formed between two nucleoside triphosphate( NTP) substrates, “de novo initiation, ” or between a 2- to ~4-nt oligoribonucleotide primer and an incoming NTP, “primer-dependent initiation, ” PDI. Although PDI had been long known to occur during transcription reactions performed in vitro( reviewed in[3]), PDI has only recently been shown to occur during the stationary phase of growth in Escherichia coli[4, 5]. In addition, the extent of PDI relative to de novo initiation at a given promoter in vivo can influence the overall abundance of transcripts produced from the promoter as well as the sequence and phosphorylation state of the 5′ ends of transcripts produced from the promoter[3–6]. To detect PDI in vivo we developed the experimental pipeline shown in Fig 1[5] that is based on two experimental considerations. First, studies of RNA metabolism in bacteria indicate that 2- to ~4-nt oligoribonucleotides( species that are sometimes referred to as “nanoRNAs”) are degraded in cells by specialized ribonucleases termed “oligoribonucleases( oligoRNase) ” or “nanoRNases”[7–9]. Thus, by increasing the concentration of an oligoRNase in vivo we decrease the concentrations of 2- to ~4-nt oligoribonucleotides in vivo( Fig 1A). Second, in vitro analyses indicate that 2- to 4-nt oligoribonucleotides effectively compete with NTPs for use as transcription primers provided the 5′ end of the RNA is complementary to sequences between positions −3 and +1( where +1 is the position of de novo initiation) and the 3′ end is complementary to positions +1, +2 or +3[6, 10–15]. Thus, PDI with 2- to 4-nt oligoribonucleotides in vitro leads to the generation of transcripts emanating from template position +1 or template positions upstream of +1( −3, −2, or −1). To unambiguously distinguish transcripts generated by PDI from those generated by de novo initiation we use high-throughput sequencing of RNA 5′ ends( 5′ RNA-seq)[16] to first, identify the primary de novo start sites associated with promoters genome-wide, and second, identify transcripts that emanate from template positions upstream of these primary de novo start sites whose abundance decreases upon ectopic expression of an oligoRNase( Fig 1B and 1C). Our 5′ RNA-seq procedure facilitates the analysis of both the sequence and phosphorylation state of the portion of the transcriptome comprising the 5′ ends of RNAs. Because transcripts generated by de novo initiation carry a 5′ triphosphate the analysis of transcripts that carry a triphosphate group can be used to identify primary de novo start sites( designated position +1), each with its associated “start site region”( i. e. positions −3 to +4)( Fig 1C, left histogram). Next, we use the analysis of the 5′ ends of all transcripts( i. e. those carrying a 5′-triphosphate, 5′-monophosphate, or 5′-hydroxyl) to determine the effect of ectopic expression of an oligoRNase on the fraction of transcripts initiated from positions upstream of +1 within each start site region( Fig 1C, middle and right histograms). The inclusion of 5′-monophosphate- and 5′-hydroxyl-containing transcripts in the analysis allows us to identify PDI events that involve a primer carrying either a 5′-monophosphate or a 5′-hydroxyl. Using this experimental pipeline we established that PDI occurs in E. coli and is growth phase-dependent[4, 5]. Specifically, we found that PDI is detected during stationary phase but is not detected during exponential phase. In addition, we found that the growth phase-dependent PDI detected in E. coli leads to a significant increase in the stationary phase expression of at least two genes, bhsA and tomB[5]. Although PDI has been shown to occur in E. coli, the impact of PDI on E. coli physiology, and the extent to which PDI occurs in other bacteria is unknown. Thus, determining the full scope of PDI in E. coli and other bacteria, the mechanisms allowing or restricting PDI in E. coli and other bacteria, the specificity with which PDI is targeted to specific promoters in E. coli and other bacteria, and the role that PDI plays in cell growth in E. coli and other bacteria are significant open questions. Here we present evidence that, in E. coli, the PDI-dependent increase in bhsA expression that occurs during stationary phase contributes to biofilm formation. Thus, our findings illuminate a previously undocumented physiological role for PDI in E. coli as a regulatory mechanism involved in biofilm growth. In addition, using a modified version of our experimental pipeline that employs a more sensitive 5′ RNA-seq protocol, we document the occurrence of PDI in the pathogenic Gammaproteobacteria Vibrio cholerae. We further find that the pattern of PDI observed in V. cholerae is strikingly similar to that observed in E. coli. Specifically, PDI in both V. cholerae and E. coli is detected during stationary phase, is not detected during exponential phase, and is preferentially targeted to promoters carrying start site regions with the sequence T−1A+1 or G−1G+1( where position +1 corresponds to the position of de novo initiation). PDI from promoters carrying T−1A+1 and G−1G+1 start site regions produces full-length transcripts emanating from position −1 that carry a 5′ hydroxyl and begin with the sequence 5′-UA and 5′-GG, respectively. Thus, the primers used to generate these transcripts must themselves begin with the sequence 5′-UA and 5′-GG, respectively. We propose that a growth phase-dependent process that preferentially generates 5′-UA and 5′-GG oligoribonucleotides occurs in both E. coli and V. cholerae.
Discussion:
The results described here, coupled with our prior analyses[5, 6], support an emerging model that PDI may be widespread in bacteria and that the extent of PDI relative to de novo initiation fluctuates as a function of growth state. During the growth conditions used in these studies we find that ~1% of transcripts generated from a given start site region in V. cholerae and ~7% of transcripts generated from a given start site region in E. coli are produced by PDI in late stationary phase( Figs 3A and 4A). However, the strict criteria we have used to measure PDI, which does not consider transcripts emanating from position +1 of a given start site region, and which considers only RNA transcripts emanating from start site regions that also produce products of de novo initiation, likely underestimates the full extent of PDI. In addition, the physiological triggers for PDI are unknown( see below). Thus, the particular growth conditions we have selected for our studies may not represent conditions where PDI is most prevalent. In this regard, over 50% of transcripts generated from a given start site region are produced by PDI in P. aeruginosa cells artificially depleted of the endogenous oligoRNase[6]. The PDI we have observed in E. coli and V. cholerae is detected in late stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying T−1A+1 and G−1G+1 start site regions. 5′ RNA-seq and primer extension analyses indicate that PDI from T−1A+1 and G−1G+1 start site regions produces transcripts emanating from position −1. Furthermore, the 5′ RNA-seq analysis indicates that transcripts emanating from position −1 of T−1A+1 and G−1G+1 start site regions begin with the sequences 5′-UA and 5′-GG, respectively. It follows that the primers used to generate these transcripts also begin with the sequence 5′-UA and 5′-GG, respectively. We therefore propose that the growth phase-dependent PDI observed in both E. coli and V. cholerae is the result of a process that leads to the generation of RNAs beginning with the sequence 5′-UA and 5′-GG at some stage during the transition from exponential phase to late stationary phase. Among all possible 2- to 4-nt RNAs beginning with 5′-UA and 5′-GG, only the linear dinucleotides 5′-UA-3′ and 5′-GG-3′ have the requisite template specificity to function as a primer during initiation at all T−1A+1 and G−1G+1 start site regions, respectively. Thus, the simplest model is that the linear dinucleotides 5′-UA-3′ and 5′-GG-3′ account for most, if not all, of the PDI observed from T−1A+1 and G−1G+1 start site regions. Our 5′ RNA-seq analyses of sequence determinants that favor PDI considered only start site regions with an A or G at position +1. This was done to ensure that our conclusions were based upon the composite behavior of start site region sequences within the context of a significant number of distinct promoters. The finding that T−1A+1 and G−1G+1 start site regions are preferentially targeted among those that contain an A or G at +1 suggests that 2- to ~4-nt RNAs beginning with 5′-UA and 5′-GG are preferentially generated in cells relative to 2- to ~4-nt RNAs beginning with the sequence 5′-AA, 5′-CA, 5′-GA, 5′-AG, 5′-CG, or 5′-UG. However, our 5′ RNA-seq analysis of individual promoters in V. cholerae also detected oligoRNase-sensitive transcripts that began with the sequence 5′-UC( a promoter associated with VCA0982)( S1 Table). Furthermore, our 5′ RNA-seq analysis of individual promoters in E. coli detected oligoRNase-sensitive transcripts that began with the sequence 5′-CC( promoters associated with ycjX, rplU, and iclR) and 5′-UU( a promoter associated with rpoS)( S7 Table). Thus, in addition to RNAs beginning with the sequence 5′-UA and 5′-GG, other 2- to 4-nt RNA species may be present at concentrations sufficient to enable PDI in V. cholerae and E. coli. We do not know the source of the oligoribonucleotides used as primers in V. cholerae and E. coli. In addition, we do not know whether or not the oligoribonucleotide primers are generated by a common mechanism in both organisms. One possibility is that oligoribonucleotides beginning with the sequence 5′-UA and 5′-GG are generated during RNA degradation. Another possibility is that the linear dinucleotides 5′-UA-3′ and 5′-GG-3′ are generated through a specific enzymatic process that awaits identification. In this regard, one intriguing speculation is that enzymes involved in the synthesis and/or breakdown of cyclic dinucleotides[27, 28, 30–34] may also have the ability to generate the linear dinucleotides 5′-UA-3′ and 5′-GG-3′, and this activity may become prevalent during stationary phase. Therefore, the cellular response to fluctuations in cyclic dinucleotide concentrations may also involve alterations in the extent of PDI. Furthermore, in principle, PDI may serve to control the intracellular concentrations of linear dinucleotides such as 5′-GG-3′ and 5′-UA-3′ by acting as a “sink” that removes such species from cells. Accordingly, PDI’s impact on cell physiology may extend beyond its effect on the composition of the transcriptome.
Primer-dependent transcription initiation , PDI , refers to an alternative mechanism of transcription initiation whereby the first phosphodiester bond within the nascent RNA is formed between a 2- to ~4-nt RNA primer and an incoming nucleoside triphosphate . Although PDI has been shown to occur in E . coli , the impact of PDI on E . coli physiology , and the extent to which PDI occurs in other bacteria is unknown . Here we establish that PDI modulates the ability of E . coli to form biofilms , a surface attached community of bacteria encased in a polymeric matrix . We further describe a significantly improved RNA-seq based method for the detection of PDI in cells . Using this method we document the occurrence of PDI in the pathogenic bacterium Vibrio cholerae . We further show that the pattern of PDI in V . cholerae is identical to that observed in E . coli , suggesting that PDI in these two organisms may occur through a conserved process that produces identical populations of 2- to ~4-nt RNA primers . Our findings suggest PDI may be widespread in Gammaproteobacteria .