Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

Abstract

During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.

The RNA Polymerase II C-terminal Domain-interacting Domain of Yeast Nrd1 Contributes to the Choice of Termination Pathway and Couples to RNA Processing by the Nuclear Exosome

“…Furthermore, genes were excluded when their TSS was downstream or over 200 nt away from the annotated start codon, when their pA site was upstream or over 200 nt away from the annotated stop codon, or when neighboring genes were less than 200 nt away. Of the resulting 1,786 genes, only 1,140 genes with higher mRNA levels than the median expression level of all yeast genes (39) were further considered for gene-averaged profiles. Among 77 snoRNA genes in the yeast genome, 51 snoRNAs, which are monocistronic and independently transcribed, were selected for analysis.…”

Uniform transitions of the general RNA polymerase II transcription complex

“…Spt6 entered early, during the 5′ transition, suggesting a recruitment mechanism independent of CTD Ser2 phosphorylation. To investigate this, we determined the ChIP-chip profile of a variant of Spt6 lacking its CTD-binding C-terminal domain (Spt6ΔC), using a yeast strain that expresses only a truncated Spt6 lacking the last 202 residues 16 . Deletion of the Spt6 CTD-binding domain led to much less recruitment of Spt6 but did not abolish its entry during the 5′ transition ( Fig.…”

“…To investigate whether the general elongation complexes are active on most genes, we correlated averaged Rpb3 and elongation factor occupancies with mRNA levels 16 . The mRNA level should be proportional to the mRNA synthesis rate of a single elongation complex times its occupancy, divided by the mRNA decay rate (see legend of Fig.…”

“…The results also show that CTD phosphorylation patterns previously observed at individual genes occur globally and that levels of CTD phosphorylation do not genome 12 . To average Pol II profiles over genes, we examined the 50% most highly expressed genes 16 that were at least 200 nt away from neighboring genes These were sorted into four main length classes, scaled to adjust for length differences and aligned by their TSS and pA sites (Online Methods). The pA site marks the point of RNA 3′ cleavage and polyadenylation, but transcription continues beyond this site until termination.…”

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling

“…Although neither Dictyostelium nor fungi possess TKs, they do have PTPs, thus suggesting that tyrosine phosphorylation arose in a common ancestor of fungi and Amoebozoa, mediated perhaps by dualspecificity kinases and regulated by dephosphorylation via PTPs (2). There are no phosphotyrosine-binding SH2 domains in fungi, but there is a sequence-related domain within the SPT6 protein that binds phospho-serine and that may have been ancestral to modern SH2 domains (5). In the fungi, therefore, phosphorylation of tyrosine appears to be used solely as a direct modulator of enzymatic function, but in Dictyostelium there are 13 SH2 domain proteins of widely varying function (6).…”