By creating an Spt4/Spt5NGN fusion protein we have been able to determine the structure of S. cerevisiae Spt4 bound to the NGN domain of Spt5. The Spt5 NGN domain bears remarkable similarities to its bacterial counterparts, even though bacteria lack an Spt4 homologue. Spt4 is structurally similar to RpoE″, which was previously suggested to be an Spt4 homologue and we further demonstrate that archaeal NusG binds to RpoE″. The homology between Spt4 and RpoE″ is largely limited to the region of Spt4 that contacts Spt5 in the Spt4-Spt5NGN structure, suggesting that the intermolecular contacts we observed are not crystal-packing artifacts and further indicating that the basic architecture of the Spt4-Spt5 complex is conserved in archaea.
What is the function of Spt4? In yeast, SPT4
is not essential for viability(Malone et al., 1993
). However, there is a near complete overlap between spt4Δ
phenotypes and partial loss-of-function mutations in SPT5
, and spt4
mutations exhibit strong genetic interactions(Hartzog et al., 1998
; Swanson et al., 1991
; Swanson and Winston, 1992
). Although it is possible that, in the intact Spt4-Spt5/RNAPII elongation complex, the zinc finger contacts DNA or RNA, we have not detected any nucleic acid binding by recombinant Spt4 alone (G.A.H. and Berra Yazar, unpublished data). In both yeast ( and data not shown) and in extracts of human cells (Kim et al., 2003
), most, if not all, Spt4 is bound to Spt5. These data support the idea that Spt4 is important for and functions entirely in the context of the Spt4-Spt5 complex.
One potential role for Spt4 is as a regulator of Spt5 conformation. Although our structure does not include the KOW domains of Spt5, several structures of NusG proteins with their single KOW domains have been solved(Knowlton et al., 2003
; Reay et al., 2004
; Steiner et al., 2002
). The KOW domain adopts a variety of orientations in these structures, indicating a highly flexible linkage between the NGN and KOW domains. A functional conformation of the NGN and KOW domains might be achieved by interaction with other transcription factors. For example, NusG only stably binds the E.coli
RNAP elongation complex in the presence of rho protein(Nehrke and Platt, 1994
). In eukaryotes and archaea it is possible that the binding of Spt4 or RpoE″ restricts the spatial relationships of the NGN and KOW domains. Structural superimposition of Spt4-Spt5NGN and NusG structures suggest the observed conformations of the KOW domain in NusG are compatible with the placement of Spt4 on the Spt5NGN (). Moreover, in one of these superimpositions, the loop between NusG's NGN and KOW domains travels near a deep groove on the surface of Spt4, placing the NusG KOW domain close to the opposite side (α2 helix) of Spt4 (). Thus, it is possible that this groove in Spt4 helps position Spt5's KOW domains relative to the Spt4-Spt5NGN core. Although few sequence changes of spt4
mutations have been reported, our structure will provide a framework for designing and interpreting effects of mutations affecting the core of the Spt4-Spt5 complex. Future challenges in studies of Spt4-Spt5 function will be to obtain structures of complexes that include the KOW domains and to understand the dynamic relationships between these domains and the core of the complex.
Evolution of the Spt4-Spt5 Complex
Spt5-Glu338, which is critical for Spt4 binding, is conserved in all eukaryotic Spt5NGN and archaeal NusG proteins (). Curiously, even though bacteria lack an apparent Spt4 homologue, many bacterial NusG and RfaH proteins have a glutamate or glutamine at a position homologous to Glu338 of Spt5 (). The fact that these proteins are widely spread and deeply branched in the bacterial lineage argues against the idea that this conservation is due to convergent evolution, which usually shows a more sporadic pattern(Barton et al., 2007
). Thus, the last common ancestor of life likely had a Glu or Gln at the same position ().
Several models may explain the conservation of a Glu/Gln at this position in bacterial proteins where Spt4 is not present. First, it is possible that the evolutionary ancestor of bacteria had an Spt4/RpoE″ progenitor which was subsequently lost; second, this Glu/Gln may be important for the structure of the NGN domain; third, bacterial NusG proteins may have an alternate conformational state in which residue 338 is important. Interestingly, bacterial NGN domains have an insertion in the NGN domain which creates a β sandwich or hairpin that is incompatible with a protein binding in the same fashion as Spt4 does to the Spt5 NGN domain (Figure S2
), suggesting a correlation between the presence of this insertion and the absence of Spt4 gene in bacteria ().
Among the RNAP II elongation control co-factors, Spt4-Spt5 is probably the oldest(Peterlin and Price, 2006
). Several observations suggest that Spt4-Spt5, archaeal NusG-RpoE″ and bacterial NusG proteins are ancient proteins involved in a core function of transcription. First, NGN proteins are ubiquitous in all branches of life and are conserved at the sequence and structural levels (). Second, in both bacteria and eukaryotes, they are implicated in the regulation of transcription elongation and related events, such as RNA processing and transcription termination. Furthermore, Spt4-Spt5 associates with RNA Pol I and rRNA genes and an spt4
mutation causes a modest decrease in growth and rRNA synthesis rates, indicating a potential role for Spt4-Spt5 in transcription elongation by RNA Pol I and rRNA processing(Schneider et al., 2006
). Similar observations have been made for the multi-subunit DNA-dependent RNA polymerases: they are found in all organisms, they share sequence, structural homology and employ similar catalytic mechanisms(Vassylyev et al., 2007
). It is most reasonable to propose therefore, that Spt4-Spt5, NusG-RpoE″ and bacterial NusG proteins share a core set of functions and mode of interacting with elongating RNA polymerases that were present in the last common ancestor of life, forming the basal transcription elongation apparatus with the primordial RNAP.