RNA polymerases (RNAPs) are core components of the cellular transcriptional machinery. Progress with functional studies of eukaryotic RNAPs has been delayed by the fact that it has not yet been possible to assemble active enzymes from individual subunits. Archaeal RNAPs are directly comparable to eukaryotic RNAPII in terms of primary sequence homology and quaternary structure. Here we report the successful in vitro assembly of a recombinant archaeal RNAP from purified subunits. The recombinant enzyme displays full activity in transcription assays and is capable, in the presence of two other basal factors, of promoter-specific transcription. The assembly of mutant enzymes yielded several unexpected insights into the structural and functional contributions of various subunits toward overall RNAP activity.
A comprehensive understanding of the molecular mechanisms operating within pro-and eukaryotic basal transcriptional machineries is a formidable intellectual goal. The combination of biochemical, genetic, and structural approaches to study the functions of basal factors and RNA polymerases (RNAPs) has started to provide very detailed insights into some of these events (reviewed in references 6 and 33). These efforts have more recently been boosted by the development of unique experimental tools based on archaeal model systems. Archaea are prokaryotes, but they contain a simplified transcriptional apparatus that is very similar to the eukaryotic core RNAPII system (reviewed in references 4 and 37). This machinery consists of TATA-binding protein (TBP), TFB (a homolog of eukaryotic TFIIB), TFE (a homolog of the eukaryotic TFIIE␣ subunit), and RNAP (homologous to eukaryotic RNAPII). Archaeal TBPs, like their eukaryotic counterparts, are responsible for the recognition of TATA elements that are located approximately 20 to 30 nucleotides upstream of the transcription start site. The DNA-bound TBP is subsequently recognized by TFB, which stabilizes the TBP/DNA complex and often enhances the sequence specificity of promoter recognition by making additional contacts with the B-recognition element (32, 40) located next to the TATA element. This TBP/TFB complex is then capable of recruiting RNAPs for promoter-specific transcription. TFE stimulates transcription from suboptimal promoters but is not strictly essential for in vitro transcription (5, 22). Unlike eukaryotic systems, no other basal factors (such as TFIIA, TFIIF, and TFIIH) are required for transcript initiation/promoter escape, and none of these steps are dependent on nucleoside triphosphate (NTP) hydrolysis to induce specific conformational changes.The entire transcriptional machinery derived from the hyperthermophilic archaeon Methanocaldococcus jannaschii has recently been reconstituted in recombinant form (47). The recombinant system is fully responsive to stimulation by transcriptional activators and thus faithfully mimics all known functions of archaeal transcription systems (36). In this study we sought to investigate the molecular mechanism governing the early steps of RNAP recruitment and transcription initiation, with special emphasis on the functional interplay of TFB and TFE with RNAP. The results reveal previously undocumented interactions between the basal factors TFB and TFE with RNAP that occur mostly at the postrecruitment stage and culminate in a carefully orchestrated modulation of core RNAP functions. Our data also demonstrate a surprising degree of redundancy of the archaeal N-terminal zinc ribbon of TFB, a novel transcription stimulatory role of the archaeal B-finger domain and a role for TFE in promoter melting and template loading. All these processes have the potential to influence key aspects of transcript initiation and promoter escape and therefore have implications for our understanding of core RNAP functions and gene expression mech...
B Br ri id dg ge e h he el li ix x a an nd d t tr ri ig gg ge er r l lo oo op p p pe er rt tu ur rb ba at ti io on ns s g ge en ne er ra at te e s su up pe er ra ac ct ti iv ve e R RN NA A p po ol ly ym me er ra as se es s A Ab bs st tr ra ac ct t B Ba ac ck kg gr ro ou un nd d: : Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available. R Re es su ul lt ts s: : Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A′ subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop. C Co on nc cl lu us si io on ns s: : The results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis.B Ba ac ck kg gr ro ou un nd d RNA polymerases (RNAPs) are central components of the cellular transcriptional machineries that are targeted by numerous regulatory proteins to fine-tune the expression of genomes in a highly controlled manner. It is therefore important to study the functional properties of RNAPs in order to understand how these are modulated during the various stages of the transcription cycle.Combined insights from biochemical, genetic and structural studies have led to the unambiguous identification of several structural motifs that participate in the key enzymatic
The TFIID complex consists of the TATA-binding protein (TBP) and associated factors (TAFs) serving to mediate transcriptional activation by promoter-specific regulators. Here we report the cloning of Drosophila TAFII250 and the assembly of a partial complex containing recombinant TBP, TAFII110 and the C-terminal domain of TAFII250. This triple complex supports Sp1 activation and reveals specific interactions between TAFII250, TBP and TAFII110.
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