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Prokaryotic Transcription Regulation by the Nascent RNA Elements

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Prokaryotic Transcription Regulation by the Nascent RNA Elements pISSN 2288-6982 l eISSN 2288-7105 Biodesign https://doi.org/10.34184/kssb.2020.8.2.33 MINI REVIEW P 33-40 Prokaryotic transcription regulation by the nascent RNA elements Seungha Hwang†, Jimin Lee† and Jin Young Kang* Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea *Correspondence: [email protected] †These authors contributed equally to this work. Transcription regulation by cis-acting elements such as DNA and RNA has not been investigated much compared to that of trans-acting elements like transcription factors because most cis-elements are much larger and more flexible than protein factors. Consequently, it was challenging to recapitulate the function of cis-elements in a reduced system for in vitro assays. However, the recent cryo-electron microscopy (cryo-EM) made it possible to study the effect of nascent RNA elements to the transcription in combination with biochemical experiments as cryo-EM does not require crystallization and tolerates heterogeneity to an extent. In this review, we briefly described the current model on prokaryotic transcriptional pausing based on the crystal and cryo-EM structures of RNA polymerases in different contexts, including an RNA hairpin pause. We then introduced two other nascent RNA elements that modulate transcription – preQ1 riboswitch and HK022 put RNA. Understanding the function of the RNA elements to transcription will deepen our understanding of the fundamental mechanism transcription and provide the structural basis for drug discovery as well as bioresearch tool development. INTRODUCTION synthesis is initiated (Tomsic et al., 2001). As the nascent RNA Transcription is an essential cellular process that transfers the is elongated, the 3’-end of the transcript makes clashes with a genetic information engraved in DNA to RNA transcripts to make loop (named “σ finger”) from the σ factor. The clashes between a template for proteins or perform various cellular regulatory the σ finger and the nascent RNA result in either the release of functions. Transcription is tightly and delicately regulated to the short nascent RNA from the RNAP (abortive initiation) or the maintain cell homeostasis and respond to the external stimulus. release of the σ factor (promoter escape), leading the complex to According to the sources of regulation, the transcriptional the elongation stage. The promoter escape efficiency is known regulators are primarily classified into trans-acting and cis-acting to be dependent on the promoter’s sequence and environmental elements. Trans-acting elements are mostly protein factors that conditions such as salt concentration (Saecker et al., 2011). Once closely interact with the transcription complexes and have effects the RNAP complex gets into the elongation step, RNA synthesis by binding to cis-acting elements or other trans-acting elements. is processive and continues until a terminator sequence on Meanwhile, cis-acting elements include sequence-specific or the DNA. At the terminator sequence, the elongation complex structure-specific DNA templates and nascent RNAs, which are disassembles to the core enzyme, the genomic DNA, and the physically connected to the RNA polymerases (RNAPs), thereby RNA transcript (Ray-Soni et al., 2016). While the elongation is the intrinsically located close to the transcription complex. Both the most processive and efficient step in the transcription cycle, the trans-elements and the cis-elements adjust transcription activity transcription rate during the elongation is not constant. Instead, it by interacting with the RNAP either directly or indirectly. occasionally pauses (about once in 100 base pairs in E.coli), and A DNA-dependent RNA polymerase, a central enzyme of some of the pauses prolong for physiological purposes such as transcription, is a multi-subunit protein complex, well conserved RNA secondary structure formation, transcription factor binding, in all three domains of life. Prokaryotic RNAP contains five and transcription termination (Kang et al., 2019). subunits (two α, β, β’, and ω) (Figure 1A) and eukaryotic In this review, we will briefly summarize the current view RNAP has 10-12 subunits whose core subunits resemble on the mechanism of transcription pausing (in particular, in those of prokaryotic RNAP (Cramer, 2002). In a transcription the prokaryotic system) and discuss three different nascent cycle, RNAPs undergo three stages - initiation, elongation, RNA elements that modulate transcription. First, we will see and termination (Figure 1B). In the initiation, an RNAP forms how a hairpin structure of nascent RNA induces transcription a holoenzyme by binding to a transcription initiation factor, σ pausing based on the recently reported cryo-EM structure of factor, binds to the promoter sequence of DNA and becomes a his pause elongation complex. Second, we will take a look “closed complex” (Gross et al., 1998). Once the promoter DNA at the working mechanism of the preQ1-I riboswitch. PreQ1-I duplex unwinds and the template DNA strand is located in the riboswitch modulates the expression of queuosine biosynthetic active site of the RNAP forming an “open complex,” the RNA enzymes according to the cellular concentration of preQ1, a www.bdjn.org Bio Design l Vol.8 l No.2 l Jun 30, 2020 33 A nascent RNA-mediated transcription regulation FIGURE 2 I The schemes of the nucleotide addition cycle. (A) At the active site of RNAP, bridge helix (BH), trigger loop (TL), and RNA-DNA hybrid form FIGURE 1 I Prokaryotic RNAP structure and its transcription cycle. (A) Pro- the active site of the RNAP with a catalytic magnesium ion. (B) A substrate karyotic RNAP consists of five subunits - two , , ’ and (The RNAP model α β β ω NTP binds to the active site, base pairing with a base from a template DNA is from PDB: 6ALF). (B) In the transcription cycle, an RNAP core enzyme and a strand. (C) The NTP binding induces TL folding into TH (trigger helix), re- -factor first form a holoenzyme and bind to a promoter sequence of template σ arranging the active site for the catalysis. (D) A phosphodiester bond forms DNA, forming the transcription bubble. Then, RNA synthesis is initiated between the bound substrate and the nascent RNA. The RNA-DNA hybrid in the presence of substrate nucleotides (initiation). As the nascent RNA translocates to vacate the active site for the next nucleotide addition cycle. prolongs, the -factor is released from the holoenzyme, letting the nascent σ This figure is modified from a figure published in (Kang et al., 2019). RNA elongate further (promoter escape), or short RNAs are released and the holoenzyme restarts the RNA synthesis (abortive initiation). After promoter escape, the formed elongation complex adds nucleotides to the nascent RNA processively (elongation) until it reaches the terminator sequence. At the terminator, the elongation complex disassembles, and the transcription cycle ends (termination). precursor molecule of queuosine. We will discuss the structures of the preQ1-I riboswitch in the presence/absence of preQ1 and the recently discovered transcriptional pausing occurring at the site close to the aptamer region of the riboswitch. Third, we will review on another RNA element HK022 put. Put is ~ 70-nucleotide (nt) long RNA element discovered in a lambdoid virus HK022 and has anti-pausing and anti-termination activities without an additional protein factor. We will cover the structures of put RNA, and the working model of the anti-termination and anti-pausing activities suggested based on the previous studies. FIGURE 3 I Schematic diagram of transcriptional pauses. In the on- pathway, RNAP translocates from “pre-translocated state” to “post- translocated state” to add the next nucleotide to the nascent RNA. During TRANSCRIPTIONAL PAUSING IN PROKARYOTES this translocation, RNAP can isomerize into an off-pathway conformation In order to add one nucleotide to the 3’-end of a nascent RNA, that blocks catalysis for a few seconds in response to the nucleic acid sequence interacting with the enzyme. This isomerized state of an elongation a substrate nucleoside triphosphate (NTP) first needs to bind to complex (EC) is termed the “elemental pause.” Elemental paused EC can the active site of an RNAP, base pairing with the template DNA further isomerize into longer-lived paused states, backtrack and RNA hairpin- strand (Figure 2A and 2B) (Kang et al., 2019). The NTP binding stabilized paused states. The domain colored in pink displays ‘swivel module’ (details are in the text). This figure is modified from (Kang et al., 2019). triggers the folding of a loop, named trigger loop, into two helices named trigger helix, making triple helices bundle forming the active site (Figure 2C). This conformational change leads to the pause can be stabilized to longer pauses by either RNA hairpin phosphodiester bond formation between the nascent RNA and formation (RNA hairpin pause) or by the backward movement the incoming substrate in the active site (Figure 2D). Then, the of the RNAP (backtrack pause). In an RNA hairpin pause, a RNA-DNA hybrid translocates one nucleotide forward to vacate short RNA hairpin with a four base pair stem structure is formed the active site for the next nucleotide addition cycle (Figure 2A within the RNA exit channel, and the formation of an RNA hairpin and 2D). interferes with the RNA synthesis at the active site of the RNAP Transcriptional pausing is thought to occur during RNA-DNA for a few minutes. In a backtrack pause, unstable or mismatched hybrid translocation step in the nucleotide addition cycles (Figure RNA-DNA hybrid induces backward movement of the RNAP, 3) (Artsimovitch and Landick, 2000). The initial pausing, named locating the 3’-end of the nascent RNA in the secondary ‘elemental pause’ occurs according to the DNA sequences at the channel, a narrow channel in RNAPs for NTPs to approach to upstream and downstream edges of the transcription bubble, the active site. The intruded RNA strand needs to be cleaved by and happens about once per 100 bases in the E.coli genome protein factors such as GreA/B, or phosphorolysis to resume the (Larson et al., 2014; Vvedenskaya et al., 2014).
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