The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.T ranscription by RNA polymerase (RNAP) II is a regulated process that requires over 60 different accessory factors that interact dynamically with the polymerase as it proceeds through the three main stages of transcription: initiation, elongation, and termination (1). Each RNAP II complex is structurally and biochemically tuned to cope with a set of conditions (e.g., the chromatin state) and to accomplish specific functions (e.g., promoter-dependent initiation) associated with a particular stage of transcription. In eukaryotes, the processes of RNAP II elongation and termination interact closely with pathways of pre-mRNA processing, 3=-end formation, and RNA export (2-4) and thus form temporally and spatially coupled activities that together determine transcriptional responses to intrinsic and extrinsic signaling and regulate RNA metabolism. The heterodimeric complex of Spt4-Spt5 is one of several protein factors that participate in all of the steps that follow transcription initiation. Saccharomyces cerevisiae Spt4-Spt5 is known to coordinate transcription elongation with chromatin remodeling and histone modification; it functions as a general elongation factor for both RNAP II and RNAP I and coordinates with 5= capping, splicing, and 3=-end processing of transcripts (reviewed by Hartzog and Fu [5]). The metazoan homolog of Spt4-Spt5, DSIF, partners with NELF and additional RNAP IIassociated complexes (e.g., P-TEFb and Mediator) to impart a pause-and-release mechanism that regulates RNAP II activity during the initiation-to-elongation transition near promoterproximal regions (6-8). While facilitative for transcription elongation, Spt4-Spt5 also pl...