RNA Polymerase

RNA polymerases (RNAP) are found in all organisms and structural studies reveal universal conservation of architecture and active site mechanism throughout evolution (Cramer et al., 2008;

From: Advances in Cancer Research , 2012

RNA Polymerase

J. Parker , in Encyclopedia of Genetics, 2001

Bacterial RNA Polymerases

Bacteria have a single cellular RNA polymerase (RNAP), whose 'holoenzyme' form has five subunits: two copies of the relatively small α-subunit (each about 36  kDa), one copy each of large β- and β′-subunits (151   kDa and 155   kDa, respectively), and one copy of the σ-subunit, also called the 'sigma factor.' The 'core' enzyme, of about 400   kDa, contains all the subunits except σ and can carry out the elongation reaction of polymerization using a DNA template and the four substrates ATP, CTP, GTP, and UTP. The evolutionarily conserved subunits are those that make up the core. However, site-specific initiation requires the σ subunit, which allows RNAP to recognize the promoter. Most bacteria encode several alternative σ factors (Escherichia coli encodes seven, Bacillus subtilis encodes 17), which may vary widely in size and which allow the RNAP to recognize several different types (sequences) of promoters. If there are several different σ factors in a cell, there must be several different holoenzymes and, therefore, one could say there are several different RNAPs in a given bacterium. However, this would be misleading, because the σ factor (of whatever kind) is only bound to the enzyme during initiation. Also, in a given bacterium, the majority of genes typically require only a single species of sigma factor and, therefore, one form of the holoenzyme predominates. In E. coli the primary σ factor, and the first discovered, has a mass of 70   kDa and is often referred to as σ70.

Initiation of transcription by RNAP at the promoter is a complex process involving many different steps. First, of course, the core enzyme must bind the appropriate σ factor. The holoenzyme then binds to promoter DNA upstream of the transcriptional start site. RNAP then interacts with the DNA, leading to melting of about 14   bp of the promoter DNA, including the transcriptional start site. There is also a conformational change of the RNAP during this process. RNAP can then begin RNA synthesis, but chain elongation often aborts, yielding short chains of less than 10 nucleotides. However, RNAP remains at the promoter and can undergo further rounds of abortive synthesis or true elongation. If the chain reaches about 10 nucleotides in length, σ factor is released and the core RNAP begins moving along the DNA template, synthesizing the RNA chain. The antibiotic rifampicin specifically inhibits initiation by bacterial RNAP, at the first or second phosphodiester bond. The antibiotic binds to the β-subunit, and resistant mutants have mutations in the gene encoding this subunit. After initiation the σ-subunit is released form RNAP and the elongation phase begins.

Elongation by bacterial RNAP is inhibited by the antibiotic streptolydigin, which also binds to the β-subunit. During initiation the RNAP may span 70–90   bp of DNA (some of which is wrapped around the enzyme), but this is reduced to about 35   bp during elongation. The newly synthesized RNA forms base pairs with the DNA template for approximately 8 or 9 nucleotides. The newly synthesized chain exits the RNAP through a channel. The rate of elongation of an RNA chain in vivo may be about 50 nucleotides per second, but this rate is the mean of rapid elongation over some sequences and pauses at others. The elongating complex is quite stable (RNA molecules of over 10   000 nucleotides may be synthesized), but the RNAP also terminates at specific DNA sequences, termed 'transcription terminators.' Some such sequences can be recognized by the RNAP itself, but others require specific accessory proteins, called 'termination factors.'

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Transcriptional Regulation

O. Amster-Choder , in Encyclopedia of Microbiology (Third Edition), 2009

Transcriptional Slippage

RNAP usually synthesizes RNA transcripts that are precisely complementary to the DNA template. However, in rare circumstances, RNAP can undergo transcriptional slippage that results in the synthesis of a transcript that is either longer or shorter than the sequence encoded by the DNA template. Such a slippage appears to occur when the polymerase transcribes homopolymeric runs. It has been proposed that the generation of transcripts that are shorter than the encoding template is due to translocation of RNAP without the incorporation of nucleotides, whereas the longer products are due to RNAP-incorporating nucleotides without translocation. Transcriptional slippage can occur during both the initiation and the elongation phases. However, the minimal length of the consecutive template nucleotides that can promote slippage in the two phases is different. During initiation, homopolymeric runs as short as 2 or 3   nt can be reiteratively transcribed by RNAP. During elongation, RNAP tends to slip only on longer runs, but the precise requirements have not been elucidated. In one case, slippage by the E. coli RNAP during elongation was reported to require runs of at least 10   dA or dT nucleotides, whereas runs of dG at the same length did not result in slippage. In some cases, the ability to slip seems to require a transcriptional pause in addition to the homopolymeric run. Transcriptional slippage is sometimes an important means of regulating transcription. It has been reported to play an important role in the regulation of transcription initiation at several bacterial operons, for example, pyrBI.

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Multi-Subunit RNA Polymerases Book I

Zachary F. Burton , in Evolution Since Coding, 2018

Abstract

Multi-subunit RNA polymerases of the two double-Ψ–β-barrel type are among the most beautiful, complex, and dynamic proteins in the human biosphere. Furthermore, multi-subunit RNA polymerases, their general transcription factors, and promoters form the core of the narrative of evolution of life on earth. In this chapter, I use a bacterial RNA polymerase–initiating complex (PDB 4XLN) interacting with promoter DNA to describe some of the features of these essential enzymes. So, this chapter is an attempt to look under the hood and partly disassemble the perplexing RNA polymerase motor. The RNA polymerase structure I selected is from Thermus thermophilus, a bacterial hyperthermophile. A bacterial RNA polymerase was selected because it is slightly simpler than archaeal and eukaryotic RNA polymerases in subunit structure and has fewer zinc (Zn) atoms. Otherwise, because of evolution, features of bacterial RNA polymerase are also features of archaeal and eukaryotic RNA polymerases.

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Polymerase

M.M. Hingorani , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

The Replisome

Polymerases responsible for genomic DNA synthesis often function as part of dynamic multiprotein assemblies known as replisomes ( Figure 1 (c)). For example, the prokaryotic E. coli replisome comprises (1) a hexameric helicase DnaB that unwinds DNA, (2) single-stranded DNA-binding (SSB) protein that coats and protects the template, (3) a primase DnaG that associates with DnaB and periodically synthesizes 10–12 nucleotide RNA primers at specific sites, and (4) the clamp loader complex that catalyzes assembly of (5) circular clamps onto the primer–template junction where they bind and tether (6) DNA polymerase III molecules to DNA during replication; the clamp loader also serves as an organizational center for replisomal proteins by binding SSB, DnaB, and two or three copies of DNA polymerase III. Association with a clamp enables a DNA polymerase to replicate several thousand nucleotides without dissociating from the template; therefore, the clamp and clamp loader are essential accessory proteins for efficient DNA replication and are highly conserved through evolution. Clamps also play a central role in coordinating access of various polymerases and other DNA metabolic proteins to specific sites on DNA such as the primer–template junction. Eukaryotic replisomes are very similar in composition, except they have more than one replicative polymerase; for example, in humans polymerase ε appears primarily responsible for leading strand and polymerase δ for lagging strand synthesis.

The crystal structures of many individual replisomal proteins have been solved; however, much remains to be discovered about the dynamic interactions of these proteins with each other and DNA. Moreover, the mechanisms of action of many of these proteins are also not well understood and are subject to intense investigation both in vitro and in vivo.

RNA Polymerases

RNA polymerases transcribe the information in DNA into RNA molecules that have a variety of functions, including messenger RNA, transfer RNA, ribosomal RNA (for protein synthesis), ribozymes (for catalysis), and microRNA (for regulation of gene expression). RNA polymerases bind specific transcription initiation sites on DNA, known as promoters, and unwind a short segment of the double helix to initiate de novo RNA synthesis on the exposed template. In this 'open' complex, RNA polymerase synthesizes short RNAs that may be aborted or elongated further into complete transcripts. Transcription proceeds in the 5′–3′ direction until it encounters a terminator sequence that disrupts the protein–nucleic acid complex and halts RNA synthesis. The catalytic mechanism of RNA polymerases is very similar to that of DNA polymerases, with Mg2+ ions enabling nucleophilic attack by the 3′-OH of the RNA primer onto the incoming rNTP, followed by release of the pyrophosphate product. Beyond these basic similarities, however, the process of RNA synthesis is very different from DNA synthesis and is subject to elaborate regulation, since only a small portion of the total genome of an organism has to be transcribed in a given cell at a time.

Bacteria typically have single RNA polymerases, but eukaryotes contain multiple RNA polymerases that are specialized for synthesis of different types of RNAs; for example, RNA polymerase II (RNAP II) synthesizes messenger RNAs and microRNAs, while RNAP I synthesizes mostly ribosomal RNAs. RNAP II is a giant protein complex made up of dozens of proteins, including the multisubunit polymerase as well as general transcription factors (e.g., TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that enable the polymerase to recognize promoters and initiate transcription. Yet other protein factors enhance or repress transcription, while other proteins enable the polymerase to elongate RNA through DNA bound by nucleosomes and packaged in chromosomes. The structure and function of RNA polymerases and their accessory proteins are subject to active investigation in order to understand how transcription occurs in an appropriate manner for optimal development and functioning of complex organisms.

Ongoing research on DNA and RNA polymerases, which are crucial for life, continues to reveal fundamental information on the development and evolution of life on earth, thereby enriching our understanding and engagement with the biological world.

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Global Regulators of Transcription in Escherichia coli: Mechanisms of Action and Methods for Study

David C. Grainger , Stephen J.W. Busby , in Advances in Applied Microbiology, 2008

A An overview of the bacterial multi-subunit RNA polymerase

In bacteria, RNA polymerase exists in two states. One form, known as the core enzyme, can catalyze RNA synthesis but is unable to bind to promoter targets in DNA. The second form of RNA polymerase, the holoenzyme, is capable of both RNA synthesis and promoter recognition. The bacterial RNA polymerase is a multisubunit enzyme and both forms of RNA polymerase posses the α 2, β and β′, and ω subunits. The RNA polymerase holoenzyme contains an additional subunit, σ, and this is the subunit that facilitates DNA recognition. Following σ-mediated DNA binding, transcription initiation occurs, the σ subunit then dissociates from the RNA polymerase–DNA–mRNA complex and the core enzyme completes the process of gene transcription. It is estimated that there are ~5000   copies of RNA polymerase in growing Escherichia coli K-12 cells, which must be distributed between ~3000   transcription units. Thus, the cell must carefully regulate the binding of RNA polymerase across its chromosome. RNA polymerase activity can be modulated by DNA sequence elements, transcription factors, nucleoid-associated proteins, small molecules, and RNA polymerase binding proteins (shown in Fig. 4.1).

Figure 4.1. Factors effecting RNA polymerase activity in E. coli.

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Polymerase☆

B.C. Case , M.M. Hingorani , in Reference Module in Life Sciences, 2017

RNA Polymerases

RNA polymerases transcribe the information in DNA into RNA molecules that have a variety of functions, including messenger RNA (mRNA; codes for proteins), and non-coding RNAs such as transfer RNA (tRNA; transports amino acids to the ribosome for protein synthesis), ribosomal RNA (rRNA; helps catalyze protein synthesis in the ribosome), microRNA (miRNA; regulates gene expression) and ribozymes (catalysts), among others. The polymerase binds specific transcription initiation start sites on DNA known as promoters, and unwinds a short segment of the double helix to initiate de novo RNA synthesis on the exposed template. In this "open" protein–nucleic acid complex, the polymerase synthesizes short RNAs that may be aborted until a stable "elongation" complex forms that can extend RNA through to a terminator sequence where the complex is disrupted to complete transcription (bacteriophage T7 RNA polymerase structure shown in Fig. 1D). Although these enzymes evolved independently, the catalytic mechanism of RNA polymerases is similar to that of DNA polymerases, with Mg2+ ions enabling nucleophilic attack by the 3′-OH of the RNA primer on the incoming rNTP to extend the polymer one nucleotide at a time. Beyond these basic similarities, however, the process of RNA synthesis is very different from DNA synthesis and is subject to elaborate regulation, since only a small portion of the total genome of an organism is transcribed in a given cell at a time.

Bacteria typically have one multi-subunit RNA polymerase that works with accessory proteins known as σ factors that enable it to recognize specific promoter sequences and initiate transcription. Eukaryotes have multiple RNA polymerases that are specialized for synthesis of different types of RNAs; for example, RNA polymerase II (RNAP II) makes mRNAs and miRNAs, while RNAP I makes mostly rRNAs, and RNAP III makes tRNAs and rRNAs. The most well studied RNA polymerase, RNAP II, is a giant protein complex made up of dozens of proteins, including the multi-subunit polymerase and general transcription factors (eg, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) that enable it to recognize promoters and initiate transcription. Yet other protein factors enhance or repress transcription, or enable the polymerase to elongate RNA through DNA bound by nucleosomes. The structure and function of RNA polymerases, and the myriad processes that regulate transcription remain an active area of research.

Ongoing research into DNA and RNA polymerases, which are crucial catalysts for life, continues to reveal fundamental information about the emergence and evolution of life on earth, thereby enriching our understanding and engagement with the biological world.

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Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods

Jeffrey A. Dewey , Bryan C. Dickinson , in Methods in Enzymology, 2020

4 Summary

The split RNAP biosensor is a powerful tool to study multiple PPIs simultaneously in live cells. In particular, split RNAP tags are capable of studying changes in several PPIs over time depending on cellular environment and PPI competition. The most important consideration when deploying the split RNAP that isn't common to other protein fragment complementation assays is its requirement for a DNA template. Considering its limitations, we believe the split RNAP biosensor can spearhead a thorough PPI network analysis within cell nuclei, specifically when investigating PPI inhibition and competition. More critically, due to its versatility and robust RNA output, the proximity-dependent split RNAP technology can be used for applications beyond just detection of PPIs, including synthetic biology applications to engineer gene circuits for cell control, selecting, and evolution.

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Single Molecule Studies of Nucleic Acid Enzymes

Samir M. Hamdan , Antoine M. van Oijen , in Single Molecule Biology, 2009

RNA Polymerases

RNA polymerase is the enzyme that reads out a DNA sequence and transcribes it into RNA. Found in all forms of life, it functions by locally melting the double-stranded DNA template and translocating along the DNA while polymerizing ribonucleotides in a template-directed fashion. The molecular mechanisms underlying transcription elongation have been studied for many decades, but numerous details remain unclear. The Escherichia coli RNA polymerase represents one of the first nucleic acid enzymes whose activity was studied at the single molecule level. Early experiments relied on the observation of the position of a small bead attached to the enzyme with the enzyme bound to a surface-tethered DNA substrate. From the amplitude of the Brownian motion of the bead, the length of the tether (i.e., the distance between the particle and the anchor point of the DNA on the surface) can be determined with an accuracy of 100   bp (Dohoney and Gelles, 2001; Schafer et al., 1991). An analysis of the particle motion allows for a direct observation of the position of the enzyme along the substrate while it synthesizes an RNA copy of the DNA template. A main advantage of this so-called tethered particle motion technique lies in its multiplexed nature. By combining appropriate immobilization densities with digital image processing, several dozens of individual tethers can be observed (Tolic-Norrelykke et al., 2004).

It was shown early on that individual RNA polymerase molecules transcribe at a constant rate over more than 1000   bp of template DNA. However, different molecules in the population appear to move at different rates (Schafer et al., 1991; Tolic-Norrelykke et al., 2004; Yin et al., 1994). The observation that a purified, seemingly homogeneous, population of enzyme molecules displayed drastically different turnover rates has been repeated for a number of different systems (Craig et al., 1996; Lu et al., 1998; Maier et al., 2000; Xue and Yeung, 1995; van Oijen et al., 2003) and will be discussed later in this chapter.

Early bulk-phase biochemical studies demonstrated that the RNA polymerase pauses frequently during the synthesis of an RNA transcript (Landick, 2006). This transcriptional pausing is believed to play an important role in the regulation of gene expression, and has been studied extensively by single molecule methods (Davenport et al., 2000; Forde et al., 2002; Herbert et al., 2006; Neuman et al., 2003). Using optical tweezers, transcriptional elongation traces of individual E. coli RNA polymerases could be obtained with high spatial resolution, allowing for a detailed study of the statistics of transcriptional pausing. These studies revealed a two-tiered pause mechanism in which particular sequences in the DNA promote entry into a paused state, followed by a secondary mechanism, such as RNA hairpin formation or backtracking, stabilizing the pause (Herbert et al., 2006).

Further technical improvements of optical trapping techniques allowed observation of movements of individual RNA polymerases along their template DNA with a resolution better than a single base pair (Abbondanzieri et al., 2005; Greenleaf and Block, 2006). These ultrasensitive methods were used to demonstrate that the unitary step size of RNA polymerase is a single base pair. An investigation of the elongation rate at different forces and ribonucleotide concentrations demonstrated that the translocation mechanism of RNA polymerases is mediated by a "Brownian ratchet" mechanism, where diffusion of the RNA polymerase between its pre- and posttranslocated states is directionally rectified by the binding of an incoming ribonucleotide (Abbondanzieri et al., 2005).

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High-Density Sequencing Applications in Microbial Molecular Genetics

Irina O. Vvedenskaya , ... Bryce E. Nickels , in Methods in Enzymology, 2018

1.2 Application of MASTER to Bacterial Transcription

Bacterial RNAP is the smallest and best-characterized member of the multisubunit RNAP family (Cramer, 2002; Darst, 2001; Ebright, 2000). Thus, bacterial RNAP provides an excellent model system for defining mechanistic principles that are relevant to the function of all multisubunit RNAPs. The focus of this chapter is on the application of MASTER for studies of transcription initiation by bacterial RNAP. In particular, we describe methods suitable for analysis of the effects of promoter sequence on transcription output by Escherichia coli RNAP. These procedures can also be adapted for studies of postinitiation steps of transcription.

The bacterial RNAP core enzyme (subunit composition α2ββ′ω) can carry out nonspecific transcription initiation and transcription elongation but must associate with a σ factor, forming the RNAP holoenzyme (subunit composition α2ββ′ω σ), to carry out promoter-specific transcription (Marchetti, Malinowska, Heller, & Wuite, 2017; Ruff, Record, & Artsimovitch, 2015). σ contains determinants for sequence-specific recognition of promoter DNA and, through those determinants, targets RNAP to promoters (Feklistov, Sharon, Darst, & Gross, 2014; Paget, 2015). During promoter-specific transcription initiation the RNAP holoenzyme binds promoter DNA, unwinds ~12–16   bp of promoter DNA to form an RNAP-promoter open complex containing an unwound "transcription bubble," and selects a transcription start site (Marchetti et al., 2017; Ruff et al., 2015). RNAP remains bound to the promoter and uses a "scrunching" mechanism during the synthesis of the first ~10   nt of the transcript (Kapanidis et al., 2006; Revyakin, Liu, Ebright, & Strick, 2006). During this phase of transcription, termed "initial transcription," the RNAP-promoter initial-transcribing complex can engage in tens or hundreds of abortive cycles of synthesis and release of short RNA products. This process, termed "abortive initiation," competes with, and limits, productive initiation. During initial transcription the RNAP-promoter initial-transcribing complex can also enter into a paused state without releasing the RNA (Duchi et al., 2016; Lerner et al., 2016). This process, termed "initiation pausing," can also limit productive initiation and has been proposed to provide a regulatory checkpoint. Once the RNAP-promoter initial-transcribing complex has synthesized a product of a threshold length of ~10   nt, RNAP breaks its interactions with promoter DNA, breaks some of its interactions with σ, and enters into the elongation phase of transcription. This last step of transcription initiation is termed "promoter escape."

Each step of transcription initiation, from promoter binding to promoter escape, can be affected by multiple sequence determinants. Because each sequence determinant for each step of transcription initiation is only one of several determinants of transcription output, their quantitative significance for different promoters differs. In addition, each step of transcription initiation can be affected by reaction conditions. Thus, predicting transcription output for given promoter sequence under different reaction conditions represents an immense challenge. To address this challenge, we have developed and applied MASTER to measure transcription output for up to at least 410 (~1,000,000) individual promoter sequences in vitro and in vivo (Hochschild, 2015; Vvedenskaya et al., 2015; Winkelman et al., 2016).

Use of MASTER for studies of transcription initiation involves the construction of a promoter template library containing up to 410 (~1,000,000) barcoded sequences, production of RNA transcripts from the template library in vitro and in vivo, and analysis of transcript 5′ ends and transcript yields (Fig. 1). The sections that follow provide a detailed protocol for each of these steps.

Fig. 1

Fig. 1. Massively systematic transcript end readout, MASTER. Top: steps in the construction of a representative MASTER template library. A representative template oligo (JB1; Table 1) carrying the lacCONS-N7 promoter, a 7-nt randomized region at positions 4–10   bps downstream of the lacCONS promoter-10 element (green), and a 15-nt barcode sequence (blue) is used as template in a PCR reaction using amplification primers (s1219 and s1220, see Table 1) that introduce BglI sites. The PCR product is digested with BglI and cloned into BglI-digested pSG289 to generate a MASTER template library that contains 47 (~16,000) sequence variants at positions 4–10   bps downstream of the lacCONS-10 element. Middle: product generated by ePCR is sequenced to assign barcodes to template-sequence variants. The PCR primers shown in red (RP1 and RPI1; Table 1) carry sequences required for analysis by the Illumina sequencer. Bottom: 5′ RNA-seq analysis of RNA produced from the library in vitro and in vivo. The sequence of the barcode is used to assign the RNA to template-sequence variant, the sequence of the 5′ end is used to define the transcription start site, and the number of reads is used to measure transcript yield from each template-sequence variant.

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Polymerase

M.M. Hingorani , in Encyclopedia of Genetics, 2001

RNA Polymerases

RNA polymerases synthesize RNA polymers complementary to a DNA template, and thus transcribe information from genes into RNA. A DNA-dependent RNA polymerase binds specific initiation sites on the DNA known as promoters, and unwinds the duplex just enough to start de novo synthesis on the template. After linking the first two nucleotides together, the polymerase elongates the RNA polymer in the 5′→3′ direction as it moves on the template. Transcription ends at a terminator site on the DNA which signals the polymerase to stop RNA synthesis. The catalytic site on RNA polymerases and the mechanism of RNA polymer formation are likely similar to those observed for DNA polymerases, except for the obvious difference that RNA polymerases use rNTPs instead of dNTPs. Beyond the basic similarities, however, RNA synthesis in the cell is a highly complex and distinctly different process from synthesis of DNA.

Gene expression plays a prominent role in the correct development and functioning of an organism, therefore transcription of genetic information is a highly regulated cellular process. Regulation of gene expression can occur at initiation of transcription or during elongation of the RNA polymer. Accordingly, RNA polymerases in both prokaryotes and eukaryotes are associated with several accessory protein factors that interact with promoters and other proteins to ensure that genes are transcribed from the right sites and under the right conditions. For example, the eukaryotic RNA polymerase II uses at least six transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFII-I) as well as other enhancers or repressors when synthesizing RNA transcripts. In fact, the more complex the organism, the more elaborate the transcription machinery appears to be. Since cells in higher eukaryotes are well differentiated, only a small proportion of the total genetic information is used by any one cell type at one time, which can only happen because transcription in complex organisms is so finely controlled.

Ongoing studies of the structure and mechanism of enzymes continue to provide detailed information on how cells maintain life. Specifically, the information on DNA and RNA polymerases, which are crucial to all life forms on earth, is essential for understanding how life evolved as well as for understanding how organisms grow and replicate to propagate life.

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