Mapping of Catalytic Residues in the RNA Polymerase Active Center

Zaychikov, Evgeny; Eisenacher, Martin; Denissova, Ludmila; Kozlov, M. V.; Markovtsov, Vadim; Kashlev, Mikhail; Heumann, Hermann; Nikiforov, Vadim; Goldfarb, Alex; Mustaev, Arkady

doi:10.1126/science.273.5271.107

Cited by 159 publications

(158 citation statements)

References 25 publications

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“…Open complexes were formed with linear rrnB P1 wild-type or mutant promoter fragments 32 P-labeled at the 5′-end of the template strand. Strand cleavage was initiated by replacement of Mg 2+ in the RNAP active site by Fe 2+ and generation of hydroxyl radicals at the bound Fe 2+ , as described (39). Cleavage products were separated on 11% acrylamide-urea gels.…”

Section: Methodsmentioning

confidence: 99%

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Winkelman

Chandrangsu

Ross

et al. 2016

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

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Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe 2+ cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ. T o initiate transcription, RNA polymerase (RNAP) and promoter DNA participate in a multistep binding reaction that results in unwinding of at least one turn of DNA and placement of the template strand into the active site. Once positioned, the initiating nucleoside triphosphate and the second NTP pair with the template, and the first phosphodiester bond is catalyzed.There is considerable information about the structure of open complexes and the position of the transcription start site (TSS) for consensus promoters and engineered scaffolds. However, perfect consensus promoters are not actually found in wild-type Escherichia coli cells, and little is known about TSS selection on native promoters and about how variation in promoter sequence affects TSS position. Comparison of the TSS for natural and synthetic Eσ 70 -dependent promoters indicates that a purine 7-8 bp downstream from the last base in the -10 element is typically used for initiation, and in some cases, two or more adjacent positions in an individual promoter are used (1-3). A pyrimidine at nontemplate strand position −1 is preferred (Fig. 1A), because the corresponding purine on the template strand makes favorable stacking interactions with the initiating NTP (4). Transcripts initiating as far as 12 bp downstream from the -10 hexamer have been reported but are very uncommon (5, 6). Changes in nucleotide concentrations alter the TSS at some promoters, and t...

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Section: Methodsmentioning

confidence: 99%

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Winkelman

Chandrangsu

Ross

et al. 2016

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

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“…Asp 28 30 , and Asp 382 are directly involved in coordinating divalent metal ions. We conclude that the active site of PARN functionally and structurally resembles the active site of the 3Ј-exonuclease domain of E. coli DNA polymerase I.…”

Section: Poly(a)-specific Ribonuclease (Parn)mentioning

confidence: 99%

Identification of the Active Site of Poly(A)-specific Ribonuclease by Site-directed Mutagenesis and Fe2+-mediated Cleavage

Ren

Martı́nez

Virtanen

2002

Journal of Biological Chemistry

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Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. 2؉ binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3-exonuclease domain of Escherichia coli DNA polymerase I. Poly(A)-specific ribonuclease (PARN)1 is the only mammalian poly(A)-specific 3Ј-exoribonuclease identified and characterized thus far (1-5). It has been shown that PARN is an oligomeric, highly processive, and mRNA cap-interacting exonuclease (3, 6 -8). A reaction pathway for PARN degradation has been proposed, and key characteristics of this pathway are the release of 5Ј-AMP as the mononucleotide product and the requirement for a 3Ј located adenosine residue with a free 3Ј-hydroxyl group (5). PARN has been cloned from human and Xenopus laevis, and the PARN polypeptide is M r 74,000 in both cases (2, 9). However, the active mammalian enzyme is significantly larger due to its oligomeric structure and most likely consists of three identical subunits (3).The function of PARN in vivo is still unknown. However, compelling evidence suggests that PARN is a key nuclease involved in both mRNA decay (7) and mRNA poly(A) tail length control during X. laevis oocyte development (2, 9). The cap interacting and poly(A) degrading properties of PARN also provide evidence that PARN may play a role in controlling initiation of protein synthesis (3, 6 -8). Besides a possible role in controlling translation, the mRNA cap binding property of PARN has a direct role in stimulating PARN degradation efficiency (3, 6, 7) and in amplifying the processive mode of degradation (8). We have recently proposed a simple model for how PARN operates (8). In this model, oligomeric PARN interacts simultaneously with the mRNA 3Ј-end-located poly(A) tail and the 5Ј-end-located cap structure. It has been suggested, based on kinetic evidence, that the cap-binding site is separate from the active site of PARN (8).The amino acid sequence of PARN implies that PARN belongs to the RNase D family of nucleases (2,11,12), of which the 3Ј-exonuclease (or proofreading) domain of Escherichia coli DNA polymerase

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“…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.…”

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confidence: 99%

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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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Mapping of Catalytic Residues in the RNA Polymerase Active Center

Cited by 159 publications

References 25 publications

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Open complex scrunching before nucleotide addition accounts for the unusual transcription start site of E. coli ribosomal RNA promoters

Identification of the Active Site of Poly(A)-specific Ribonuclease by Site-directed Mutagenesis and Fe2+-mediated Cleavage

Multiple personalities of the RNA polymerase active centre

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