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

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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). This short transcription (Borukhov et al., 1993). These pauses play essential

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roles in context-dependent transcription regulation processes such as RNA secondary structure formation or transcription factor binding. his PAUSE RNA HAIRPIN RNA hairpin pauses are characterized in diverse bacteria (Landick and Yanofsky, 1984; Yakhnin et al., 2006). Among the identified RNA hairpin pauses, his pause, which is located at the leader sequence of the histidine synthesis operon of Salmonella typhimurium and adjusts the expression of the operon, is the best studied in its sequence specificity and kinetics. Since RNA hairpin pause was discovered, two different hypotheses of the pausing mechanism were suggested. The first FIGURE 4 I Conformational changes of hisPEC compared to non-paused hypothesis was a ‘rigid body model’ in that an RNAP is a rigid EC. (A) cryo-EM structure of hisPEC. The pause hairpin stem, drawn in dotted body, and the folding of the RNA hairpin pulls the RNA transcript balls, resides in the RNA exit channel. (B) Superimposition of the Cα traces of the swiveled domains in hisPEC (colored in red) and non-paused EC in order to base pair and form duplex RNA structure. As a result, (colored in gray). A red circle marks the axis of swiveling. (C) Superimposition the nascent RNA changes its conformation within the RNAP of the RNA-DNA hybrids from hisPEC (colored in magenta and yellow) and elongation complex, and the complex would become inactive. non-paused EC (colored in gray). Compared to the non-paused EC, hisPEC exhibits tilted or ‘half-translocated’ RNA-DNA hybrid, explaining reduced the This model was refuted because 1-nt insertion between the RNA transcription rate. This structure was also found in ‘elemental paused’ EC. hairpin and the RNA-DNA hybrid prolonged the pause, instead of releasing the tension of RNA pulling and decreasing the pause duration by providing more space between duplex RNA and the 4B). This swiveled conformation inhibits nucleotide addition elongation complex. The second model was an ‘allosteric model’ because SI3, one of the swiveled domains, makes clashes with β in that the RNAP changes the conformation of the active site subunit upon trigger loop folding, which is crucial for catalysis. upon the RNA hairpin formation in the RNA exit channel, which is 3) The RNA-DNA hybrid in hisPEC exhibited a tilted confor ~ 40 Å away from the active site. Corresponding to the allosteric mation termed ‘half-translocated state’ (Figure 4C). The half- model, followed cross-linking experiments, mutational studies, translocated RNA-DNA hybrid does not let the substrate and cys-pair assays showed that RNA hairpin formation in the binding to the active site because the template DNA base is not RNA exit channel induces conformational changes in the active available for a substrate, but base pairs with the last nucleotide site (Toulokhonov et al., 2001; Toulokhonov and Landick, 2003; of a nascent RNA. This half-translocated RNA-DNA hybrid was Nayak et al., 2013; Hein et al., 2014). observed in a minor population of the dataset that contains the Thanks to the recent technical advances in cryo-electron RNAP elongation complexes without an RNA hairpin in the RNA microscopy (cryo-EM), the structure of E.coli his pause exit channel. The hisPEC without a pause hairpin in the RNA exit elongation complex (hisPEC) were independently determined by channel can be thought of as an elemental paused elongation two groups (Kang et al., 2018; Guo et al., 2018). The structure complex that reflects the former state before the RNA hairpin revealed unprecedented structural changes of bacterial RNAP as forms. Therefore, the half-translocated state is suspected to follows (Figure 4): occur in the elemental pause, and the conformation is retained in 1) The RNA hairpin stem is located within the RNA exit channel, the RNA hairpin paused state. while the loop region of the pause hairpin is exposed to the From the structure and the biochemical data, we envision the solvent and not resolved in the cryo-EM structure (Figure 4A). following model for the RNA hairpin-stabilized pause. First, an The inner wall of the RNA exit channel is lined with positively elongation complex pauses at a pause site via the elemental charged amino acid residues, presumably assisting in forming an pause mechanism, providing a time window for an RNA hairpin RNA hairpin within the channel. formation in the RNA exit channel. At this stage, the RNA-DNA 2) Based on the crystal structure of Thermus aquaticus elemental hybrid assumes a half-translocated conformation. Second, paused elongation complex, the RNAP structure in an RNA the RNA hairpin formation allosterically stabilizes the swiveled hairpin paused state was predicted to have an open clamp conformer, thereby relocating SI3. The relocated SI3 inhibits conformation, in that β’ clamp domain forming the main channel proper folding of the trigger loop for the nucleotide addition of an RNAP is located more distant from β protrusion domain, and delays the transcription for minutes. Although how RNA making the main channel of the RNAP wider than that in a hairpin-induced structural rearrangement prolonged the pausing close clamp conformation (Weixlbaumer et al., 2013). However, is revealed in the cryo-EM structure, it is still unknown how in contrast to the expectation, the clamp in the hisPEC was the RNA hairpin in the RNA exit channel induces the swiveling not open but rotated in a closed position with other domains, movement. Molecular simulation aided by computation would including β’ jaw, shelf, and SI3, forming ‘swivel module’ (Figure aid in understanding how those two actions link in the RNAP.

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PreQ1-I RIBOSWITCH Riboswitches are located in the 5’-untranslated region (5’- UTR) controlling gene expression, and consist of two domains: aptamer domain and expression domain (Winkler and Breaker, 2003). The aptamer domain binds to the ligands with high affinity, and the expression domain undergoes structural changes in response to the ligand binding (Hermann and Patel, 2000). The ligands of riboswitches are diverse from salt, small molecules to environmental factors such as temperature and pH. Thus far, about 40 different classes of riboswitches have been discovered by the bioinformatics research. TPP (thiamin pyrophosphate) riboswitches are the most abundant among them, and cobalamin riboswitch, FMN (flavin mononucleotide) riboswitch, SAM FIGURE 5 I Function and structure of preQ -I riboswitch. (S-adenosylmethionine)-I riboswitch, fluoride riboswitch, purine 1 (A) Regulation of gene expression by the preQ1 riboswitch. Ligand-induced pseudoknot riboswitch, and preQ1 riboswitch are also found in multiple docking favors the formation of a terminator hairpin, leading to the species (Mironov et al., 2002; Winkler and Breaker, 2003; Baker decreased expression of the downstream genes (B) NMR structure of a preQ -I riboswitch. A compact pseudoknot with three loops and two stems et al., 2012; McCown et al., 2017). 1 encapsulates preQ1 (PDB code:2L1V; Kang et al., 2009). Among them, preQ1-I riboswitch was first identified in the 5’- UTR (untranslated region) of the ykvJKLM operon of Bacillus

Subtilis (Bsu) encoding queuosine synthesis enzymes (Barrick et preQ1-II) by the different conserved sequences, secondary al., 2004). The ligand of preQ1-I riboswitch, preQ1, is a precursor structures, and different modes of ligand recognition. Bsu preQ1-I of queuosine, a hyper-modified nucleoside occupying the riboswitch has the smallest aptamer domain (34-nt) among all anticodon wobble position of asparaginyl, tyrosyl, histidyl, and known riboswitches. According to the Rfam database, nearly aspartyl transfer RNAs (tRNAs) (Roth et al., 2007; Okada et al., 900 sequences across 647 species have been identified for

1978). When preQ1 binds to the preQ1-riboswitch, the expression preQ1-I riboswitches in the phyla Firmicutes, Proteobacteria, and of the queuosine synthesis operon located at the downstream of Fusobacteria. For preQ1-II riboswitches, 429 sequences across the preQ1-I riboswitch decreases (Roth et al., 2007). For example, 423 species have been identified in the order Lactobacillales.

Bsu preQ1-I riboswitch regulates transcription by switching the While preQ1-II riboswitch regulates only translation, preQ1-I secondary structure of the nascent RNA from the anti-terminator, riboswitches regulate either transcription or translation according an RNA hairpin that prevents transcription termination, to the to the species (Eichhorn et al., 2014). terminator, an RNA hairpin that triggers transcription termination. In 2009, three different groups independently reported

On binding preQ1 to the preQ1-I riboswitch, the anti-terminator structures of ligand-bound preQ1-I riboswitch aptamers - X-ray hairpin formation is inhibited by the pseudoknot formation. As a crystal and NMR solution structures of a transcription-regulating result, the terminator hairpin forms and the transcription halts, Bsu riboswitch bound to preQ0, and a crystal structure of blocking the expression of the queuosine synthetic enzymes translation-regulating Thermoanaerobacter tengcongensis (Tte)

(Figure 5A) (Kang et al., 2009). preQ1 riboswitch (Kang et al., 2009; Klein et al., 2009; Spitale

Prokaryotes synthesize PreQ1 de novo from GTP via a multi et al., 2009). The structures revealed that preQ1-I riboswitch enzyme pathway. First, 7-cyano-7-deazaguanine (preQ0) is folds into an H-type pseudoknot in the presence of preQ1 synthesized in a series of reactions involving GTP cyclohydrolase (Figure 5B). PreQ1-I riboswitch contains two stems (P1, P2) (GCH1), 6-carboxy-5,6,7,8-tetrahydropterin synthase (QueD), separated by three loops (L1, L2, L3), and in the presence of

7-carboxy-7-deazaguanine (CDG) synthase (QueE), and preQ0 preQ1, the 3’-end of the A-rich tail of the preQ1-I aptamer base synthase (QueC). PreQ0 is converted to preQ1 catalyzed by QueF, pairs with the center of the P1 hairpin loop to form an H-type and incorporated into tRNAs by tRNA:guanine transglycosylase pseudoknot (Figure 5). Solution and crystal structures of Bsu

(TGT) enzymes. Then QueA and QueG enzymes convert PreQ1 preQ1-I riboswitch are nearly identical with small variations. The to Q (Slany et al., 1993; Slany et al., 1994). PreQ1 riboswitches structure of the Tte preQ1-I riboswitch is also highly similar to the are only found in prokaryotes as eukaryotes do not synthesize Bsu riboswitch structures with a nearly identical binding pocket queuosine precursors. However, queuosine is used in a wide despite sequence differences, particularly in the residues above range of cellular functions in both eukaryotes and bacteria such the binding pocket. as eukaryotic cellular development and proliferation, relieving Recently, Widom et al. reported a single-molecule study hypoxic stress, neoplastic transformation, tyrosine biosynthesis, on Bsu preQ1-I riboswitch bound to the RNAP elongation and virulence of pathogenic bacteria such as Shigella flexneri complex (Widom et al., 2018). In the study, the chemically-

(Iwata-Reuyl, 2003). synthesized preQ1-I riboswitch was labeled with acceptor and

PreQ1 riboswitches are divided into two types (preQ1-I and donor fluorescent dyes at the loop region of P1 stem and after

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P2 stem, respectively, and the folding of the preQ1-I riboswitch phage, and most of them require anti-termination protein factors pseudoknot was observed by FRET (Förster Resonance Energy such as host-encoded Nus factors and phage-encoded N and Transfer) signal. The result showed that the folding was inhibited Q protein. These anti-termination factors directly or indirectly in the presence of DNA only, but recovered to the level of a bind to the RNAP, modifying the enzyme to resist transcription naked RNA folding when the RNA is in the elongation complex termination (Mogridge et al., 1995). In particular, a lambda-like binding to the template DNA and an RNAP. Furthermore, in phage, HK022, has a distinct anti-termination mechanism that the in vitro transcription assay, preQ1-I riboswitch associated utilizes a nascent RNA element without the help from additional elongation complex paused at U46 (named ‘que pause’), with a protein factors. This cis-acting anti-termination element is named pause half-life of 42 seconds in the absence of preQ1 and with a “put (polymerase utilization) RNA” (King et al., 1996). significantly shortened half-life of 15 seconds in the presence of HK022 has two put RNAs, putL and putR located after the preQ1. Although the RNA duplex in the que paused elongation viral promoters, PL and PR, respectively. Although there are complex is in the same location as the pause hairpin stem of his some differences in details such as the length of their stems pause, que pause has some distinct characteristics compared and the numbers of the bulges and the internal loops, both putL to RNA hairpin pause. First, que pause is recognized in both and putR have similar secondary structures in that two stem- Bsu and E.coli; however, Bsu does not respond to his pause loops separated by an unpaired base (King et al., 1996) (Figure at all (Artsimovitch and Landick, 2000). Second, NusA, a host 6A). Interestingly, put RNA has dual activities – anti-termination factor that stabilizes RNA hairpin pause, did not stabilize the que and anti-pausing activities (Komissarova et al., 2008). As with pause. Third, the deletion of the β flap-tip region of the RNAP the anti-termination, the anti-pausing activity requires no abolishes RNA hairpin pause while it only modulates the pause accessory protein. However, the mechanisms of anti-pausing duration in que pause. Besides, the RNA at que pause does not and anti-termination are expected to be distinct because they undergo cleavage in response to the GreB factor, implying that have different distance-dependence – the anti-pausing activity the que pause is different from the backtrack pause, either. diminishes as the distance between put RNA and the pausing From the various experiments including single-molecule site increases. In contrast, the anti-termination activity is not study, cross-linking experiment, transcription assay, and MD dependent on the distance between the terminator and put RNA simulation, Widom et al., suggested a model relating preQ1-I (Sloan et al., 2007). riboswitch and que pause as follows: (1) The presence of stalled The in vivo transcription assay using various base substitution RNAP provides a favorable free energy landscape for folding of or deletion of put RNA revealed that the activity of put RNA the nascent preQ1-I riboswitch. (2) The ligand-free but pre-folded retained when the sequence of the bases in the stem region riboswitch pseudoknot stabilizes the paused state of RNAP changes, maintaining its duplex structure. Corresponding to via interactions with the β flap-tip and with positively charged this result, the mutations with severe effects of terminator residues on β’ subunit. (3) preQ1 binding stabilizes a distinct readthrough disrupt the base pairs in the stem region (King et al., docked conformation that counteracts pausing. However, we still 1996). However, the sequence of specific residues of put RNA is do not know how the que pause influences the preQ1-I riboswitch involved in the activity. Among the unpaired bases in putL, G35, pseudoknot folding and modulates transcription. The structural study of preQ1-I riboswitch bound to the elongation complex will also expand our knowledge on the transcription regulation by the nascent RNA elements and will provide valuable information for practical purposes such as antibiotics discovery and the tool development for the biomedical research (Blount and Breaker, 2006; Jenkins et al., 2011; Wu et al., 2015).

HK022 put RNA Lambdoid phages utilize the host transcription system to express a set of viral enzymes to integrate the viral genome into the host DNA. These genes are located downstream of the transcription terminators to control the progression of viral gene expression. Therefore, to take the steps of viral infection, the disassembly of the RNA transcription complex at the terminator sites needs to be prevented. For this purpose, the transcription complex FIGURE 6 I Schematic structure of HK022 put RNA and its inactivating changes to terminator-resistant form when it encounters anti- mutation on E.coli RNAP. (A) Schematic structure of HK022 putL. (B) The terminators. This process is called anti-termination (King et al., structure of the E.coli elongation complex (PDB code: 6ALF) The β’ Y75 residue of the zinc-binding domain, located near the RNA exit channel, is 1996; Banik-Maiti et al., 1997). marked with green spheres. β’ Y75N mutation abolishes the anti-pausing and The various types of anti-termination have been studied from λ anti-termination activity of put RNA.

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located between two stem-loop, is important for the put activity RNA-bound RNAP elongation complex is necessary. However, (Figure 6A). It has a homolog in putR (G298), and substitution of the reconstitution of put RNA-associated RNAP elongation G35 to A makes severe put RNA activity defect. (Banik-Maiti et complex is very challenging because the widely-used method al., 1997). In addition, deletion or substitution of some unpaired of RNA synthesis by T7 RNAP or chemical synthesis cannot be bases at loops or bulges, such as G45, G46, A63, and A64, applied for this study. These methods do not produce functional decreases the readthrough severely, suggesting that the identity put RNA. Besides, the de novo generation of put RNA-bound of the bases at these positions is important for the activity elongation complex by adding NTP substrates to template-DNA (Banik-Maiti et al., 1997). Besides the mutations on put RNA, the bound RNAP initiation complex would make heterogeneous anti-termination activity of HK022 put RNA can be abolished by a populations of the elongation complexes with various lengths of mutation in the RNAP. The entire trends of readthrough rate with RNAs. It was suggested to prepare a homogenous population β’ subunit mutated RNAP (β’-Y75N) and the inactive put RNA of HK022 put RNA-associated elongation complex by de novo with base mutations were similar in the in vitro and in vivo assays transcription with a roadblock on the template DNA (King et al., (King et al., 1996; King et al., 2004). 2003). In the method, the lac operator sequence was inserted The anti-pausing activity of putL RNA is displayed in the U-rich in the template DNA, and lac repressors were added to bind to backtrack pausing site located in ~ 20-nt downstream of putL the sequence. As a result, an RNAP would initiate transcription RNA. This anti-pausing activity is abolished by the mutation on at the promoter in the template DNA, elongate RNA through the stem 2 (put-) (Figure 6A) or β’-Y75N RNAP (Figure 6B) (Clerget the DNA, and stall the transcription blocked by the bound lac et al., 1995; Sen et al., 2001; Komissarova et al., 2008). β’ Y75 repressor. The lac repressor was removed by adding IPTG, residue of RNAP is located in the zinc-binding domain (cys70- and the resulting elongation complex behaved in the same way cys88 of E.coli) (King et al., 2004) (Figure 6B). With β’-Y75N with the non-roadblocked elongation complex. However, this RNAP, the strength of the pause increases compared to wild type in vitro reconstitution still is very inefficient and sub-optimal for (WT) RNAP. Besides, WT RNAP protects downstream region of structural study. Therefore, for the structural study of HK022 put stem 1 and the whole stem 2 of put RNA from the RNase V1 and RNA-associated elongation complex, a novel or more optimized T1 cleavage while β’-Y75N RNAP does not protect any region of method for the complex reconstitution is required. put RNA (Sen et al., 2001). These results indicate that put RNA binds to the RNAP through the zinc-binding domain, and this CONCLUSION interaction is critical for the anti-pausing activity. Furthermore, it An RNA polymerase is a central molecule in transcription, and was shown that WT put RNA inhibited backtracking at the U-rich its modulation plays a critical role in the regulation of gene pausing site while β’-Y75N RNAP or put- mutant associated expression. The fundamental mechanism of transcription RNAP undergoes backtracking, suggesting the possibility that regulation by conformational changes of RNAPs in diverse put RNA inhibit pausing by hinder backtracking movement of the contexts just started to be revealed owing to the advances in RNAP. cryo-EM methodology as the past dominant methodology of To explain the working mechanism of put RNA, three different the structural study, X-ray crystallography, could not reflect models were suggested as follows: (1) pausing and termination the dynamic solution structures of RNAPs. From the cryo-EM are manipulated in the same mechanism. However, this option structure of the RNA hairpin-paused elongation complex, we was eliminated as anti-pausing, and anti-termination activities of learned how RNA hairpin formation in the RNA exit channel of the put RNA showed different distance dependency, as mentioned RNAP induces transcriptional pausing and gained insights on the above (Sloan et al., 2007). (2) putL RNA stem-loop structure transcription regulation by the secondary structures of nascent inhibits the backtracking by preventing re-enter of RNA into RNAs. To expand the insights and deepen our understanding of RNAP exit channel physically, and interaction between putL the fundamental mechanism of transcription regulation by cis- stem and RNAP is needed only for anti-termination. This option acting elements, atomic structure determination of the RNAP was also removed because adding oligos complementary elongation complex in association with nascent RNA elements to the RNA region upstream of the RNA exit channel to the is essential. The results of the structural studies will provide put RNA-containing elongation complex reduced the anti- fundamental knowledge on the mode of action in RNAP in pausing activity, although the upstream RNA duplex is retained the transcription regulation as well as the structural basis for (Komissarova et al., 2008). (3) putL binds to EC and generates biomedical applications such as drug development. anti-pausing and anti-termination but in different mechanisms. Unlike the second model, the secondary structure of RNA Original Submission: Jun 1, 2020 itself does not interrupt backtracking, and RNA binding to EC Revised Version Received: Jun 9, 2020 is required for functions. This model is currently favored, but Accepted: Jun 9, 2020 the details of how the secondary structure and the specific residues of put RNA play anti-pausing and anti-termination is to be elucidated. To this end, the structural study of HK022 put

38 Bio Design l Vol.8 l No.2 l Jun 30, 2020 www.bdjn.org Seungha Hwang, Jimin Lee and Jin Young Kang

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