Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase

Sosunov, Vasily V.; Sosunova, Ekaterina; Mustaev, Arkady; Bass, I. A.; Nikiforov, Vadim; Goldfarb, Alex

doi:10.1093/emboj/cdg193

Cited by 181 publications

(210 citation statements)

References 35 publications

(70 reference statements)

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“…2b). This was consistent with a two-metal ion catalytic mechanism 17,18 , but was unexpected because free Pol II only binds metal A, whereas metal B was observed previously only when a nucleoside triphosphate (NTP) was present 19,20 . The two metal ions are bridged by a rotated aspartate D481 side chain (Fig.…”

supporting

confidence: 87%

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

175

193

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The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex 1,2 indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions 3,4 enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and proteinnucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA-RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12-13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors 5 , and the bacterial initiation factor sigma has TFIIB-like topology 1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function [6][7][8] . TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.Our previous X-ray analysis of the Pol II-TFIIB complex at 4.3 Å resolution provided a partial backbone model of TFIIB 1 . To obtain a complete and atomic structure, we co-crystallized Pol II with a TFIIB variant lacking the mobile amino-terminal tail and carboxy-terminal cyclin fold (Methods, Supplementary Table and Supplementary Fig. 1). The resulting Pol II-TFIIB structure at 3.4 Å resolution provides details of the interactions of the four TFIIB domains with Pol II: the B-ribbon with the dock, the B-core N-terminal cyclin fold with the wall, the B-reader helix with the RNA exit tunnel, and the B-linker helix with the coiled-coil of the clamp (Fig. 1).The structure reveals the entire course of the TFIIB polypeptide chain through the Pol II cleft, including the previously lacking 1 B-reader loop (residues 67-79) and a new 'B-reader strand' (residues 80-83). The B-reader loop does not reach the active site, but instead interacts with the Pol II rudder and fork loop ...

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supporting

confidence: 87%

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Sainsbury

Niesser

Cramer

2012

Nature

175

193

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“…A preinsertion site for NTP entry into the prokaryotic RNAP elongation complex has been proposed in ref. 45. The position where ppGpp is found in the T. thermophilus cocrystal appears to overlap the position of the NTP entry (preinsertion) site in the structure of the yeast transcription elongation complex (46).…”

Section: Effects Of Dksa On Positive Regulation Of Amino Acid Promotementioning

confidence: 94%

DksA potentiates direct activation of amino acid promoters by ppGpp

Paul

Berkmen

Gourse

2005

Proc. Natl. Acad. Sci. U.S.A.

298

413

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Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp͞ DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.RNA polymerase ͉ rRNA transcription ͉ transcription initiation ͉ amino acid biosynthesis ͉ stringent response A mino acid starvation in Escherichia coli results in global changes in gene expression called the ''stringent response'' (reviewed in ref. 1). This regulatory response is initiated by entry of uncharged tRNAs into the A site of the ribosome and activation of the ribosome-associated RelA protein to synthesize the ''alarmone'' ppGpp (used here to refer to both guanosine 5Ј-diphosphate 3Ј-diphosphate and its pentaphosphate precursor). During starvation for amino acids, expression of stable RNA (rRNA and tRNA) is inhibited, whereas expression of enzymes for amino acid biosynthesis and transport is induced. These negative and positive responses are both relA dependent (e.g., refs. 2-6).ppGpp directly and specifically inhibits the initiation of transcription from stable RNA promoters in vivo and in vitro (7)(8)(9)(10)(11)(12). Although the details of the mechanism by which ppGpp exerts its effects on transcription initiation are still ill-defined, it has been shown that ppGpp decreases the lifetimes of competitorresistant complexes between RNA polymerase (RNAP) and all promoters that have been examined (12). The complex formed by RNAP at rRNA promoters is intrinsically short-lived, and ppGpp further decreases the lifetime of this complex. Thus, we have proposed that ppGpp shifts the equilibriu...

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“…Intrinsic 'TL+39 NMP'-catalysed hydrolysis is relatively slow and it is no surprise that evolution led to the emergence of specific factors to assist hydrolysis. Cleavage factors bind in the vicinity of the active centre, and help RNAP to chelate Mg 2+ II and, possibly, position the attacking water molecule (Laptenko et al, 2003;Sosunova et al, 2003). We investigated how the bacterial Gre factor cooperates with the 'TL+39 NMP' catalytic module to enhance the rate of the reaction.…”

Section: Replacement Of the Tl By Transcription Factors And Switchingmentioning

confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

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Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals.A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https: //www.youtube.com/watch?v=79Z7iXVEPo4 TRIGGER LOOP: A NEW CATALYTIC DOMAIN OF THE RNA POLYMERASE ACTIVE CENTRE Multi-subunit RNAPs are the enzymes that perform transcription in all living organisms. RNAPs are highly conserved, and emerged before the divergence of the bacteria and archaea/eukaryote lineages. All RNAPs share the invariantly conserved catalytic core of five subunits: b, b9, 2a and v (bacterial nomenclature is used throughout). The two largest subunits, b and b9, form the catalytic cleft where the reaction of addition of nucleoside monophosphates (NMPs) to the growing RNA takes place. As with many other nucleic-acid-managing enzymes (Steitz & Steitz, 1993) and all nucleic-acid-polymerizing enzymes (Steitz, 1998), RNAP uses a two-metal-ion (Mg 2+ ) mechanism to catalyse the phosphotransfer reaction. One of the metal ions, Mg 2+ I, is chelated by the invariant triad of aspartates of the b9 subunit, whilst the other one, Mg 2+ II, is brought by substrates, e.g. by the incoming nucleoside triphosphate (NTP). Mg 2+ I stabilizes the negative charge on the attacking oxygen of the hydroxyl group of the 39 NMP of RNA. Mg 2+ II assists the leaving of the pyrophosphate, and both metal ions ligate the non-bridging oxygen to stabilize the pentacovalent transition state. Substitutions in the aspartate triad lead to almost full inactivation of the enzyme (Zaychikov et al., 1996).The emergence of the first crystal structure of multi-subunit RNAP (Zhang et al., 1999) ignited structure-based functional analysis of the enzyme by many researchers. We were interested in the disordered loop (referred to initially as the G-loop and, later, as the TL) of the highly conserved G domain of the b9 subunit. In the structure, this disordered region was located .20 Å from Mg 2+ I. However, we found that deletion of this loop had a dramatic effect on catalysis by slowing down NMP addition~10 4 times -an effect close to that of mutations in the aspartate triad. Three years later we foun...

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Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase

Cited by 181 publications

References 35 publications

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

DksA potentiates direct activation of amino acid promoters by ppGpp

Multiple personalities of the RNA polymerase active centre

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