Biomedical subjects

M Patturajan

Publications and source records attributed to M Patturajan.

7 recordsLinked to original sources

PubMed1998

Growth-related changes in phosphorylation of yeast RNA polymerase II.

The largest subunit of RNA polymerase II contains a unique C-terminal domain (CTD) consisting of tandem repeats of the consensus heptapeptide sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Two forms of the largest subunit can be separated by SDS-polyacrylamide gel electrophoresis. The faster migrating form termed IIA contains little or no phosphate on the CTD, whereas the slower migrating II0 form is multiply phosphorylated. CTD kinases with different phosphoryl acceptor specificities are able to convert IIA to II0 in vitro, and different phosphoisomers have been identified in vivo. In this paper we report the binding specificities of a set of monoclonal antibodies that recognize different phosphoepitopes on the CTD. Monoclonal antibodies like H5 recognize phosphoserine in position 2, whereas monoclonal antibodies like H14 recognize phosphoserine in position 5. The relative abundance of these phosphoepitopes changes when growing yeast enter stationary phase or are heat-shocked. These results indicate that phosphorylation of different CTD phosphoacceptor sites are independently regulated in response to environmental signals.

Amino Acid Sequence↗

PubMed1998

A nuclear matrix protein interacts with the phosphorylated C-terminal domain of RNA polymerase II.

Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975-6980, 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.

Amino Acid Sequence↗

PubMed1997

The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II.

DNA-dependent RNA polymerases (RNApol) are Zn2+ metalloproteins where the Zn2+ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn2+ ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn2+, as well as partial activity. However, Zn2+ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn2+-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2+-native enzymes. Zn2+-reconstituted RNApolII or one Zn2+-RNApolII showed a slower abortive synthesis rate. 65Zn2+-blotting experiments indicated that the removal of one Zn2+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn2+ ions has any effect on substrate recognition, we followed the binding of (gamma-AmNS)UTP, a fluorescent substrate analog to RNApolII. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+-native RNApol binds substrate at a single site.

Binding Sites↗

PubMed1997

A CTD function linking transcription to splicing.

Since its discovery in 1985, the function of the C-terminal domain (CTD) of RNA polymerase II has been a puzzle. Recent studies suggest that the CTD functions as a linear platform for assembly of complexes that splice, cleave and polyadenylate pre-mRNA. A new set of CTD-associated SR-like proteins (CASPs) have been implicated in pre-mRNA processing and transcription elongation as a component of the emerging 'transcriptosome'.

Animals↗

PubMed1996

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

Amino Acid Sequence↗

PubMed1995

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis.

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 microM for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of alpha-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from C. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of non-specific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

Candida↗

PubMed1994

Accurate transcription initiation by RNA polymerase II from Candida utilis.

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.

Candida↗