Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5

Abstract

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...

Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest

“…We examined cross-linking within the transcription bubble (Ϫ5) and upstream DNA (Ϫ21/Ϫ23). It was proposed that the K1L1 of Spt5, together with Spt4, "cradles" upstream DNA (32). However, the results show that the K1L1 mutant cross-linked at the Ϫ5 and Ϫ21/Ϫ23 positions (Fig.…”

“…Previous models of eukaryotic and archaeal Spt4/5 suggest it may play a role in maintaining bubble integrity by aiding in the closing of the bubble as DNA exits RNA polymerase (8,10,18,32). The experiment was conducted under two conditions in the presence and absence of Spt4/5.…”

“…Activities of Spt4/5-A high resolution x-ray crystal structure of the KOW1-linker (K1L1) region of Spt5 was solved, and this fragment of the protein bound single-and double-stranded nucleic acids (32). However, the importance of this region, which is believed to be the counterpart of the KOW domain in prokaryotic Spt5 homologs, for the biochemical activities of the complex is not known.…”

“…KOW domains are generally part of larger Tudor domains that are known to interact with nucleic acids (30,31). In fact, a crystal structure of an isolated KOW1-linker (K1L1) fragment of Spt5 and biochemical analysis using isolated K1L1 provided evidence that it binds to nucleic acids (32). Furthermore, in vivo cross-linking data conducted on yeast Spt5 revealed that KOW domains 4 and 5 interact with surfaces on RNAPII, including the Rpb4/7 sub-module.…”

Transcription Regulation in Archaea

“…Archaeal Spt5, homologous to bacterially encoded NusG, consists of two domains: the NusG N-terminal (NGN) domain and a single C-terminal Kyrpides-Ouzounis-Woese (KOW) domain with affinity for single-stranded RNA (83,84,87); eukaryotic Spt5 typically contain three to six repeats of the C-terminal KOW domain (94)(95)(96). Critical, direct molecular interactions between Spt5 and RNAP have been identified in both Bacteria and Archaea (83,84,87,88,95,(97)(98)(99), and the conservation of RNAP and Spt5 infers that these same interactions are used in Eukarya.…”