scholarly journals A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthew A Sdano ◽  
James M Fulcher ◽  
Sowmiya Palani ◽  
Mahesh B Chandrasekharan ◽  
Timothy J Parnell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Sh2 Domain ◽  
Heptad Repeat ◽  
Side Chain ◽  
Phosphate Binding ◽  
Recognition Mechanism ◽  
Repressive Chromatin ◽  
Repeat Domain

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

Functional studies of the carboxy-terminal repeat domain of Drosophila RNA polymerase II in vivo.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 599-613 ◽  
Author(s):  
W J Brickey ◽  
A L Greenleaf
Keyword(s):  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Terminal Repeat ◽  
Mrna Levels ◽  
Wild Type ◽  
Functional Studies ◽  
Repeat Domain ◽  
Carboxy Terminal

Abstract To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity.

scholarly journals A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II

1998 ◽  
Vol 18 (4) ◽  
pp. 2406-2415 ◽  
Author(s):  
Meera Patturajan ◽  
Xiangyun Wei ◽  
Ronald Berezney ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Consensus Sequence ◽  
Nuclear Speckles ◽  
Repeat Domain ◽  
Active Transcription

ABSTRACT 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.

scholarly journals Genetic Interactions of Spt4-Spt5 and TFIIS With the RNA Polymerase II CTD and CTD Modifying Enzymes in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 487-497 ◽  
Author(s):  
Derek L Lindstrom ◽  
Grant A Hartzog
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Normal Function ◽  
Genetic Interactions ◽  
Elongation Factors ◽  
Repeat Domain ◽  
Ctd Phosphatase ◽  
Rna Polymerase Ii Ctd

Abstract Genetic and biochemical studies have identified many factors thought to be important for transcription elongation. We investigated relationships between three classes of these factors: (1) transcription elongation factors Spt4-Spt5, TFIIS, and Spt16; (2) the C-terminal heptapeptide repeat domain (CTD) of RNA polymerase II; and (3) protein kinases that phosphorylate the CTD and a phosphatase that dephosphorylates it. We observe that spt4 and spt5 mutations cause strong synthetic phenotypes in combination with mutations that shorten or alter the composition of the CTD; affect the Kin28, Bur1, or Ctk1 CTD kinases; and affect the CTD phosphatase Fcp1. We show that Spt5 co-immunoprecipitates with RNA polymerase II that has either a hyper- or a hypophosphorylated CTD. Furthermore, mutation of the CTD or of CTD modifying enzymes does not affect the ability of Spt5 to bind RNA polymerase II. We find a similar set of genetic interactions between the CTD, CTD modifying enzymes, and TFIIS. In contrast, an spt16 mutation did not show these interactions. These results suggest that the CTD plays a key role in modulating elongation in vivo and that at least a subset of elongation factors are dependent upon the CTD for their normal function.

scholarly journals Identification of a binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain of RNA polymerase II.

1996 ◽  
Vol 16 (7) ◽  
pp. 3361-3369 ◽  
Author(s):  
R Baskaran ◽  
G G Chiang ◽  
J Y Wang
Keyword(s):  
Tyrosine Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Sh2 Domain ◽  
Glutathione S Transferase ◽  
Transient Overexpression ◽  
Rnap Ii

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD) in vitro. Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. We show here that a CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes the Km 0.4 microM to increase by 2 orders of magnitude. Direct binding of the CTD-ID to CTD and to RNAP II could be demonstrated in vitro. Phosphorylation of a recombinant glutathione S-transferase-CTD by c-Abl was observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylated nor associated with the glutathione S-transferase-CTD in vivo. Transient overexpression of c-Abl also led to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II could be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II was again dependent on the presence of the CTD-ID in Abl. Finally, the ability of c-Abl to phosphorylate and associate with RNAP II could be correlated with the enhancement of transcription by c-Abl in transient cotransfection assays. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo.

scholarly journals The Two Steps of Poly(A)-Dependent Termination, Pausing and Release, Can Be Uncoupled by Truncation of the RNA Polymerase II Carboxyl-Terminal Repeat Domain

2004 ◽  
Vol 24 (10) ◽  
pp. 4092-4103 ◽  
Author(s):  
Noh Jin Park ◽  
David C. Tsao ◽  
Harold G. Martinson
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
The Body ◽  
Terminal Repeat ◽  
Repeat Domain ◽  
Carboxyl Terminal

ABSTRACT The carboxyl-terminal repeat domain (CTD) of RNA polymerase II is thought to help coordinate events during RNA metabolism. The mammalian CTD consists of 52 imperfectly repeated heptads followed by 10 additional residues at the C terminus. The CTD is required for cleavage and polyadenylation in vitro. We studied poly(A)-dependent termination in vivo using CTD truncation mutants. Poly(A)-dependent termination occurs in two steps, pause and release. We found that the CTD is required for release, the first 25 heptads being sufficient. Neither the final 10 amino acids nor the variant heptads of the second half of the CTD were required. No part of the CTD was required for poly(A)-dependent pausing—the poly(A) signal could communicate directly with the body of the polymerase. By removing the CTD, pausing could be observed without being obscured by release. Poly(A)-dependent pausing appeared to operate by slowing down the polymerase, such as by down-regulation of a positive elongation factor. Although the first 25 heptads supported undiminished poly(A)-dependent termination, they did not efficiently support events near the promoter involved in abortive elongation. However, the second half of the CTD, including the final 10 amino acids, was sufficient for these functions.

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