Abstract
Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive cross-linker, we mapped interactions between Spt5 and RNAP II in Saccharomyces cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.
Highlights
RNA polymerases (RNAPs), which carry out transcription in all living organisms, are highly conserved at the level of sequence, structure, function and molecular mechanisms [1]
To determine if eukaryotic Spt5 interacts with RNA polymerase II (RNAP II) in a similar way, we used an in vivo site-specific cross-linking technique [32,33]
Note that two proteins cross-linked at different sites may migrate differently on the gel. These results indicate that Spt5 interacts with the clamp, protrusion and wall domains of RNAP II in yeast
Summary
RNA polymerases (RNAPs), which carry out transcription in all living organisms, are highly conserved at the level of sequence, structure, function and molecular mechanisms [1]. An RNAP interacts with different factors during transcription initiation and elongation [1]. NusG/Spt family proteins are universally conserved transcription elongation factors that play pivotal roles in transcription and transcription related processes by binding to RNAP and interacting with other transcriptionassociated factors [4]. Eukaryotic Spt proteins are much larger (1063 and 1087 residues in Saccharomyces cerevisiae and humans, respectively) and more complex, consisting of an N-terminal acidic domain, an NGN domain, multiple KOW domains and a C-terminal repeat (CTR) domain [4]. The NGN domain of archaeal and eukaryotic Spt associates with Spt, a relatively small zinc-binding protein (61, 102 and 117 residues in Pyrococcus furiosus, S. cerevisiae and humans, respectively), to form a heterodimeric Spt4/5 complex
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
