Affinity purification of Spt5-associated proteins reveals a series of
related Spt5-Pol II complexes.
Derek L. Lindstrom (1),
Nemone Muster (2), Kimberly C. Wachter (1), Sharon L. Squazzo (1), John
R. Yates 3rd (2), Grant A. Hartzog (1)
(1) MCD Biology, UC Santa
Cruz, Sinsheimer Labs, Santa Cruz, CA 95064, USA; (2) Department of
Cell Biology, The Scripps Institute, La Jolla, CA 92037
The Spt4-Spt5
complex is an essential transcription elongation factor that is
conserved in eukaryotes. The mechanism by which this complex functions
is not known, but genetic and biochemical studies have shown that it can
both inhibit and promote elongation and that its function is related to
or dependent upon the CTD of RNA polymerase II (Pol II). We used
immunoaffinity purification and mass spectrometry to identify Spt5-associated proteins. These include Pol II and proteins involved in Pol
II transcription, including capping enzyme and the elongation factors
TFIIF and TFIIS. Interestingly, Spt6 and the CP complex (Spt16-Pob3-Nhp6; FACT in humans) were associated with Spt5. Like Spt5, these
proteins have been proposed to regulate transcription elongation in the
context of chromatin. We also identified and characterized a novel
transcription factor, Spt24. To further characterize Spt5-Pol II
complexes, we examined interactions between each of these factors by co-immunoprecipitation. Our data indicates that Spt5 is associated with at
least 3 distinct Pol II complexes: a Pol IIA complex that
includes Spt6 and Spt24, a Pol IIO complex that includes
these factors as well as capping enzyme, and a second Pol IIO
complex that lacks capping enzyme and Spt24, but includes Spt16-Pob3.
Genetic analysis supports the distinctions between these complexes. We
propose that Spt5 is likely to be a component of a series of temporally-related Pol II elongation complexes.
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