Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

Fresco, Lucille D.; Buratowski, Stephen

doi:10.1073/pnas.91.14.6624

Cited by 61 publications

(71 citation statements)

References 57 publications

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“…pRS314-hakin28(T17D), pRS314-hakin28(K36A), and pRS314-hakin28(T162A) will be described by Keogh et al (unpublished data). The remaining plasmids were constructed as previously described (7,9,17,33,35,44,50,52,55). DNA manipulations and transformation into bacteria were performed by standard techniques (3).…”

Section: Methodsmentioning

confidence: 99%

Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

Rodriguez

Cho

Keogh

et al. 2000

Molecular and Cellular Biology

178

145

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The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.Eukaryotic pre-mRNAs are transcribed by RNA polymerase II (Pol II) and undergo several processing events before maturing into mRNA. Soon after initiation of transcription, premRNA is capped at its 5Ј terminus (27,48). Transcripts are further processed by the splicing and polyadenylation machineries before translocation to the cytoplasm for translation. Cotranscriptional mRNA processing is facilitated by the recruitment of mRNA processing factors to the carboxy-terminal domain (CTD) of the Pol II large subunit (8,22,23,26,36,37,56).The CTD is composed of a tandemly repeated heptad with the consensus sequence YSPTSPS (1, 10). Mammalian Pol II CTD has 52 repeats, whereas the yeast Saccharomyces cerevisiae CTD has only 26 (12). Deletion of the mouse (4), Drosophila (58), or yeast (2, 43) CTD is lethal, and partial deletions result in conditional phenotypes, reducing transcription and response to activators (5,19,38,49). The CTD is phosphorylated in vivo, primarily at serine 2 and serine 5 of the heptapeptide consensus repeat (12). Hyperphosphorylation of the CTD appears to be coordinated with transcription initiation and elongation in vivo (45, 54). Phosphorylation is mediated by one or more CTD kinase activities, but the timing and role of specific kinases are not clearly defined.Several putative CTD kinases are members of the cyclindependent kinase (CDK) family. These kinases typically consist of a catalytic subunit bound to a regulatory cyclin subunit. The Kin28-Ccl1 (Cdk7-cyclin H) kinase complex associated with the general transcription factor TFIIH can phosphorylate the CTD after preinitiation complex (PIC) formation, thereby positively regulating transcription (16,21). The Srb10-Srb11 (Cdk8-cyclin C) kinase complex is associated with the RNA Pol II h...

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Section: Methodsmentioning

confidence: 99%

Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

Rodriguez

Cho

Keogh

et al. 2000

Molecular and Cellular Biology

178

145

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“…A CET1 CEG1 double disruption strain was created as follows: a 4.8-kb EcoRI/SalI fragment from pBS-LYS2 containing the LYS2 gene was subcloned into the EcoRI and XhoI sites of pBSKS(ϩ)(⌬RI-X)-CEG1 (6). The resulting plasmid [pBSKS(ϩ)(⌬RI-X)-ceg1⌬3::LYS2] releases a 5.5-kb fragment of ceg1⌬3::LYS2 with NotI/PstI digestion.…”

Section: Dna Cloning Methodsmentioning

confidence: 99%

The Essential Interaction between Yeast mRNA Capping Enzyme Subunits Is Not Required for Triphosphatase Function In Vivo

Takase

Takagi

Komarnitsky

et al. 2000

Molecular and Cellular Biology

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The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxyterminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxyterminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.Eukaryotic and viral mRNAs are modified at their 5Ј end by a cap structure which consists of a 7-methylguanosine moiety attached to the 5Ј terminus via a 5Ј-5Ј linkage. Cellular mRNA capping enzyme is a bifunctional enzyme: RNA 5Ј-triphosphatase removes the ␥-phosphate from the 5Ј end of the RNA substrate to leave a diphosphate end, and GTP::mRNA guanylyltransferase subsequently transfers GMP from GTP to the 5Ј-diphosphate RNA end. A separate enzyme, RNA (guanine-7-)-methyltransferase, adds a methyl group to the N-7 position of the guanine cap to leave m 7 GpppN 1 -(35). Capping enzyme from Saccharomyces cerevisiae is a heterodimer of RNA triphosphatase and guanylyltransferase subunits (20) encoded by the CET1 and CEG1 genes, respectively. Both genes are essential for cell viability (34, 39). CET1 and CEG1 homologs from Schizosaccharomyces pombe and Candida albicans functionally replace the S. cerevisiae genes (36,42,44). The fungal guanylyltransferase subunits have amino acid similarity to viral and metazoan guanylyltransferases, indicating a common reaction mechanism (11, 41). In contrast to the two subunit yeast enzymes, capping enzyme from higher eukaryotes are a single polypeptide consisting of an aminoterminal RNA triphosphatase domain and a carboxy-terminal guanylyltransferase domain. Both mouse (MCE or MCE1) and human (HCE or HCE1) enzymes can replace CEG1 and CET1 in vivo (16,17,24,43,47). Interestingly, the higher eukaryotic RNA triphosphatase domains belong to the PTP (protein tyrosine phosphatase) superfamily (27, 38, 47) and do not resemble the fungal phosphatases (39, 44).Cellular capping enzymes ...

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“…This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7)(8)(9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates.…”

mentioning

confidence: 97%

A ribozyme that ligates RNA to protein

Baskerville

Bartel

2002

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

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We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5 terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzymenucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.in vitro selection ͉ nucleic acid tag ͉ phosphoamide ͉ Tat ͉ TAR I n vitro selection has been used in combination with rational design to identify and optimize numerous ribozymes and binding motifs (1-3). The results of these experiments continue to give insight into the capacity of RNA and DNA to catalyze a variety of chemical reactions and the ability of nucleic acids to recognize an array of substrates. Some of these catalysts are being developed into effective tools for use in molecular biology. These include allosteric ribozymes, used as detectors of small molecules and proteins, and DNA enzymes that efficiently cleave target sequences (4-6).Here we use in vitro selection coupled with rational design to produce a new ribozyme that ligates RNA to protein through the formation of a phosphoamide bond. This is similar to a reaction found in naturally occurring proteins such as guanyltransferase, and DNA and RNA ligases that form phosphoamide bonds as enzyme-nucleotidyl intermediates during cap formation and ligation events (7-9). The selection of this new ribozyme expands the repertoire of RNA-catalyzed chemistry and further illustrates the ability of RNA to chemically modify a variety of substrates. Moreover, this ribozyme might be used to synthesize oligonucleotide-tagged proteins in vitro and in vivo. These attached oligonucleotides might be useful as affinity tags or could function as amplifiable identifying markers. For example, this ribozyme might be used to attach mRNAs to the proteins that they encode. A diverse library of mRNA-protein fusions could be used in conjunction with in vitro selection strategies to identify previously undescribed proteins with unique or enhanced properties, as has been shown previously by using phage display, ribosome display, and mRNA display (10-13). Materials and MethodsPool Construction. The double-stranded DNA pool was constructed from two 110-nt DNA fragments, each containing 76 completely randomized positions (14). Each fragment was amplified separately in 110 ml PCR reactions and ligated through BanI sites to generate a final pool with the sequence: TTCTAATACGACTCACTATAG-GACAGCTCCGAGCATTCTCGTGT...

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Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

Abstract: Nasent mRNA chains are capped at the 5' end by the addiion ofa guanylyl residue to form a G(5')ppp (5'

Cited by 61 publications

References 57 publications

Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II

The Essential Interaction between Yeast mRNA Capping Enzyme Subunits Is Not Required for Triphosphatase Function In Vivo

A ribozyme that ligates RNA to protein

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