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Jack Greenblatt

Publications and source records attributed to Jack Greenblatt.

24 records · Page 2Linked to original sources
PubMed2003

Solution structure of the carboxyl-terminal domain of RAP74 and NMR characterization of the FCP1-binding sites of RAP74 and human TFIIB.

FCP1 (TFIIF-associated CTD phosphatase) is the only known phosphatase specific for the phosphorylated CTD of RNAP II. The phosphatase activity of FCP1 is strongly enhanced by the carboxyl-terminal domain of RAP74 (cterRAP74, residues 436-517), and this stimulatory effect of TFIIF can be blocked by TFIIB. It has been shown that cterRAP74 and the core domain of hTFIIB (TFIIBc, residues 112-316) directly interact with the carboxyl-terminal domain of hFCP1 (cterFCP, residues 879-961), and these interactions may be responsible for the regulatory activities of TFIIF and TFIIB on FCP1. We have determined the NMR solution structure of human cterRAP74, and we have used NMR methods to map the cterFCP-binding sites for both cterRAP74 and human TFIIB. We show that cterFCP binds to a groove of cterRAP74 between alpha-helices H2 and H3, without affecting the secondary structure of cterRAP74. We also show that cterFCP binds to a groove of TFIIBc between alpha-helices D1 and E1 in the first cyclin repeat. We find that the cterFCP-binding site of TFIIBc is very similar to the binding site for the HSV transcriptional activator protein VP16 on the first cyclin repeat of TFIIBc. The cterFCP-binding sites of both RAP74 and TFIIBc form shallow grooves on the protein surface, and they are both rich in hydrophobic and positively charged amino acid residues. These results provide new information about the recognition of acidic-rich activation domains involved in transcriptional regulation, and provide insights into how TFIIF and TFIIB regulate the FCP1 phosphatase activity in vivo.

Binding Sites↗
PubMed2003

Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II.

Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Chromatin↗
PubMed2002

Regulation of transcription elongation by phosphorylation.

The synthesis of mRNA by RNA polymerase II (RNAPII) is a multistep process that is regulated by different mechanisms. One important aspect of transcriptional regulation is phosphorylation of components of the transcription apparatus. The phosphorylation state of RNAPII carboxy-terminal domain (CTD) is controlled by a variety of protein kinases and at least one protein phosphatase. We discuss emerging genetic and biochemical evidence that points to a role of these factors not only in transcription initiation but also in elongation and possibly termination. In addition, we review phosphorylation events involving some of the general transcription factors (GTFs) and other regulatory proteins. As an interesting example, we describe the modulation of transcription associated kinases and phosphatase by the HIV Tat protein. We focus on bringing together recent findings and propose a revised model for the RNAPII phosphorylation cycle.

Amino Acid Sequence↗
PubMed2002

Evidence that the KH RNA-binding domains influence the action of the E. coli NusA protein.

The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. An essential function in E. coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of lambda. Tandem KH domains have been identified downstream of the S1 domain. We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains. The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action. The changes in both KH domains interfere with NusA binding to RNA. A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action. However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein. These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the lambda N protein.

Amino Acid Substitution↗
PubMed2002

Bridging structural biology and genomics: assessing protein interaction data with known complexes.

Currently, there is a major effort to map protein-protein interactions on a genome-wide scale. The utility of the resulting interaction networks will depend on the reliability of the experimental methods and the coverage of the approaches. Known macromolecular complexes provide a defined and objective set of protein interactions with which to compare biochemical and genetic data for validation. Here, we show that a significant fraction of the protein-protein interactions in genome-wide datasets, as well as many of the individual interactions reported in the literature, are inconsistent with the known 3D structures of three recent complexes (RNA polymerase II, Arp2/3 and the proteasome). Furthermore, comparison among genome-wide datasets, and between them and a larger (but less well resolved) group of 174 complexes, also shows marked inconsistencies. Finally, individual interaction datasets, being inherently noisy, are best used when integrated together, and we show how simple Bayesian approaches can combine them, significantly decreasing error rate.

Actin-Related Protein 3↗