Filipovska, K. their substrates and products. It is becoming clear that many LACE1 antibody RNA-processing reactions occur during transcription elongation (reviewed in references 34 and 45). However, our understanding of the elongation phase of transcription is incomplete. In vitro, two general transcription elongation factors, TFIIF and TFIIS, are sufficient to stimulate in vivo rates of elongation on naked DNA templates (25). In contrast, elongation on nucleosome-bound templates is inefficient, even in the presence of TFIIF and TFIIS, suggesting a requirement for other factors (9, 25, 26). Several factors have been implicated in the regulation of transcription elongation through chromatin. Among these is the yeast Spt4-Spt5 complex, known as DSIF in human cells (21, 57). DSIF/Spt4-Spt5 can inhibit and promote elongation of RNA polymerase II (Pol II) on cellular genes and is required for the stimulation of transcription elongation by human immunodeficiency virus type E3 ligase Ligand 9 1 Tat in vitro (24, 28, 57, 64). A second elongation factor, Spt6, interacts genetically with (2, 27, 44). Finally, genetic and biochemical studies of Spt4, Spt5, and Spt6 in yeast have led to the proposal that they function by affecting chromatin structure (6, 51). A third protein complex, FACT, composed of the human Spt16 and SSRP1 proteins, promotes elongation by Pol II through nucleosomes in vitro (40, 41). Its yeast homolog, E3 ligase Ligand 9 the CP (or SPN) complex, is composed of two tightly associated subunits, Pob3 and Cdc68/Spt16 (the name Cdc68 will be used here to avoid confusion of Spt6 with Spt16), as well as a weakly associated HMG box protein Nhp6 (7, 8, 18). Mutations in lead to similar mutant phenotypes, and these genes also display numerous genetic interactions with each other (reviewed in references 22 and 60). Thus, although direct evidence is lacking, the overlapping genetic and biochemical behaviors of these Spt proteins suggest that they may collaborate to carry out a common or overlapping set of functions in vivo. Recent observations suggest a functional interplay between Spt4-Spt5 and the C-terminal heptapeptide repeats (CTD) of Pol II. The CTD serves as a scaffold for factors involved in transcription and processing. For example, the mRNA capping enzyme, polyadenylation factors, and certain splicing proteins all associate with the CTD of transcribing RNA Pol II. Furthermore, perturbation of the CTD or addition of CTD peptides affects splicing in vitro and in vivo, suggesting that the CTD may affect the efficiency of processing reactions (reviewed in references 34 and 45). Biochemical studies show that P-TEFb, a CTD kinase that regulates elongation, works in conjunction with DSIF and possibly FACT (56, 58). In addition, we have recently shown that and display an extensive set of genetic interactions with the CTD and enzymes that modify the CTD’s phosphorylation status, including protein kinases similar to P-TEFb (31, 39). Finally, the human and Spt5 proteins interact with the capping enzyme (43, 59). These studies show that Spt4-Spt5 is a candidate for an elongation regulator that mediates interactions between the elongating polymerase and processing events linked to the CTD. A mechanistic understanding of Spt4-Spt5 function requires a knowledge of the proteins that associate with this complex. Here we describe affinity purification of Spt5 from yeast extracts. Using mass spectrometry, E3 ligase Ligand 9 we identified a large number of proteins that copurified with Spt5. Many of these interactions were subsequently verified by coimmunoprecipitation and genetic analysis. We show that Spt5 associates with Pol II and the general elongation factors TFIIF and TFIIS, as well as with Spt6, Cdc68, and Pob3. Furthermore, we demonstrate that Spt5 coimmunopurifies with the yeast capping enzyme and cap methyltransferase and that and mutations cause splicing defects in yeast. In addition, we show that Spt5 copurifies and genetically interacts with a recently identified Spt6-interacting protein, Iws1. Through extensive coimmunoprecipitation analyses we provide evidence that Spt5 participates in at least three different protein complexes with Pol II. These observations provide new evidence of close connections.