Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1-Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.longation by RNA polymerase II (RNAPII) involves a number of factors that interact directly or indirectly with the enzyme (1). Among these RNAPII-interacting proteins in the budding yeast Saccharomyces cerevisiae are the RNA chain cleavage factor TFIIS, the chromatin assembly factor Spt16/Pob3 (known as FACT in higher eukaryotes), the polymerase-associated factor (PAF) complex, and Spt4/Spt5, which is orthologous to DRB sensitivityinducing factor (DSIF) in humans. These factors can associate with the transcription complex from the promoter-proximal region to the cleavage and polyadenylation signal or the transcription termination site. In higher eukaryotes, DSIF and the negative elongation factor (NELF), which appears not to exist in S. cerevisiae, restrain elongation by RNAPII soon after initiation (2). As a consequence, many inactive genes in higher eukaryotes have ''paused'' or ''poised'' RNAPII molecules downstream of their promoters (3).An important event during elongation by RNAPII is phosphorylation of the heptapeptide repeats (consensus sequence YSPTSPS) in the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII (4). Phosphorylation on S5 of the CTD repeats is catalyzed by the Kin28 subunit (CDK7 in humans) of the general initiation factor TFIIH. S5 phosphorylation tends to decline across yeast transcription units (5, 6) and is reversed by the Ssu72 subunit (7) of the cleavage an...

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