Like the core RNAP subunits, Spt5 is present in all three kingdoms of life and is one of the most ancient components of the transcription machinery (8
). In particular, the NGN domain of Spt5 shows striking structural similarity between bacteria, archaea and eukaryotes. We show here that this similarity extends to the mechanisms that Spt5 uses to bind RNAP and to promote transcription. We provide evidence that a conserved interaction between the tip of the RNAP clamp coiled-coil and a hydrophobic depression on the Spt5 NGN domain mediates complex formation and elongation stimulation.
We have identified three hydrophobic residues (alanine-4, tyrosine-42 and leucine-44) in the archaeal Spt5 NGN domain that are apparently part of the RNAP-binding site. These hydrophobic residues line a concave depression that is located opposite of the Spt4 interaction surface and oriented away from the Spt5 KOW domain of our model of full-length Spt4/5, consistent with RNAP binding without steric hindrance. We also showed that replacement of ten amino acids forming the tip of the clamp coiled-coil motif of the largest subunit of the M. jannaschii
RNAP (A' residues 255-264) with a tetra glycine linker abolishes Spt4/5 binding and Spt4/5 elongation stimulation. Consistently, a similar mutation interferes with the response of bacterial RNAP to the NusG paralogue RfaH (21
). Also consistent, a coiled-coil fragment of the E. coli
RNAP (β′ 262–309) interacts with NusG (residues 1–132) in a genetic assay (44
), and the NusG NGN domain mediates RNAP binding and elongation stimulation (17
). The elongation-stimulatory activity of Spt4/5 and NusG are generally mild in vitro
. Whereas we measured a 1.8-fold increase in the formation of the runoff transcript, Landick and colleagues (17
) report a 1.2-fold increase, and Nudler and colleagues (42
) obtain a 1.8-fold increase in the elongation rate of bacterial RNAP.
The elongation–stimulatory mechanism of Spt4/5 is likely allosteric, since the Spt4/5 binding site on the tip of the RNAP clamp coiled-coil is located far above active centre on one rim of the polymerase cleft. Since this site is close to the path of the NTS in the elongation complex (47
), Spt4/5 could modulate elongation by altering RNAP–nucleic acid interactions, maybe by facilitating DNA–RNA strand separation or upstream DNA strand rewinding. However, our observation that Spt4/5 stimulates elongation in the absence of the NTS argues against these mechanisms. Alternatively, Spt4/5 could influence the active centre by altering the clamp position, as suggested for the bacterial NusG paralogue RfaH (21
). Details of the Spt4/5 mechanism remain to be investigated, including the mechanism of transmitting an allosteric signal, and the question whether Spt4/5 stimulates elongation at the level of transcription rate, RNAP processivity, or both. In eukaryotic transcription systems it is exceedingly difficult to directly monitor the effect of Spt4/5 on elongation by RNAPII, which require additional factors including the Paf-1 complex and Tat-SF1 (49
). Thus, the archaeal transcription systems offer a unique opportunity to unravel the molecular mechanisms of Spt4/5.
Comparison of our data with published results reveals interesting differences between archaeal and eukaryotic Spt4/5. In particular, archaeal Spt4/5 binds directly to RNAP and independently of the DNA–RNA nucleic acid scaffold, whereas human Spt4/5 (DSIF for DRB-sensitivity inducing f
actor) binding to RNAPII apparently depends on the presence of the RNA transcript (50
), although another study showed that association of Spt4/5 with elongating RNAPII was insensitive to nuclease treatment (51
). An RNA-dependency in eukaryotic systems could be due to interactions between the transcript and Spt5 domains that are not conserved in the archaeal variant, including the additional copies of KOW domain and C-terminal repeats. Alternatively, the interactions between the hyperthermophilic archaeal Spt4/5 and RNAP may be stronger owing to more stringent requirements for the stability of RNAP–transcription factor complexes at the biologically relevant high reaction temperatures of M. jannaschii
. Similarly, the interactions between RNAP subunits in M. jannaschii
are stronger when compared to S. cerevisiae
RNAPII subunits (23
Whereas the N-terminal NGN domain of NusG/Spt5 mediates a conserved RNAP contact, the C-terminal KOW domain(s) in these factors apparently mediate additional, species-specific interactions. In the bacterial system, an activity of NusG during transcription termination is dependent on both NGN and KOW domains, but especially the latter has been implicated in facilitating the recruitment and/or interactions with accessory factors that include Rho, NusA, NusB and NusE (17
). Eukaryotic Spt5 harbours multiple copies of the KOW domain that may mediate some of the multiple physical and functional interactions of Spt4/5, including interactions with the negative transcription elongation factor NELF, kinases that regulate NELF, Spt5 and RNAPII by phosphorylation, including P-TEFb and Bur-1/Bur-2, and factors that are involved in chromatin remodelling such as Spt6 and FACT, and RNA processing factors such as the mRNA capping enzyme and the cap methyl transferase (51
). Hitherto no factors have been identified that interact with the Spt5 KOW domain in the Archaea.
Our results reveal that the structure and the elongation function of Spt4/5 and NusG, including the interaction site on the distinct RNAPs, are universally conserved throughout the three domains of life. Considering that eukaryotic Spt4/5 regulates both RNAPI and II (52
), Spt4/5 seems evolutionary ancient, and the molecular mechanisms by which it regulates gene expression are likely to have their origin prior to the diversification of distinct classes of eukaryotic RNA polymerases, and indeed before the split of the three main lineages of life, Bacteria, Archaea and Eukarya. Since the transcription factors that govern transcription initiation in Bacteria and Archaea/Eukarya are not homologous, this lends itself to the hypothesis that the regulation of transcription elongation might even precede the regulation of transcription initiation.