Michael S. Bartlett

Topography of the Euryarchaeal Transcription Initiation Complex

Transcription in the Archaea is carried out by RNA polymerases and transcription factors that are highly homologous to their eukaryotic counterparts, but little is known about the structural organization of the archaeal transcription complex. To address this, transcription initiation complexes have been formed with Pyrococcus furiosus transcription factors (TBP and TFB1), RNA polymerase, and a linear DNA fragment containing a strong promoter. The arrangement of proteins from base pair -35 to +20 (relative to the transcriptional start site) has been analyzed by photochemical protein-DNA cross-linking. TBP cross-links to the TATA box and TFB1 cross-links both upstream and downstream of the TATA box, as expected, but the sites of most prominent TFB1 cross-linking are located well downstream of the TATA box, reaching as far as the start site of transcription, suggesting a role for TFB1 in initiation of transcription that extends beyond polymerase recruitment. These cross-links indicate the transcription factor orientation in the initiation complex. The pattern of cross-linking of four RNA polymerase subunits (B, A′, A″, and H) to the promoter suggests a path for promoter DNA relative to the RNA polymerase surface in this archaeal transcription initiation complex. In addition, an unidentified protein approximately the size of TBP cross-links to the non-transcribed DNA strand near the upstream edge of the transcription bubble. Cross-linking is specific to the polymerase-containing initiation complex and requires the gdh promoter TATA box. The location of this protein suggests that it, like TFB1, could also have a role in transcription initiation following RNA polymerase recruitment.

Transcription Factor E Is a Part of Transcription Elongation Complexes

A homologue of the N-terminal domain of the α subunit of the general eukaryotic transcription factor TFE is encoded in the genomes of all sequenced archaea, but the position of archaeal TFE in transcription complexes has not yet been defined. We show here that TFE binds nonspecifically to single-stranded DNA, and photochemical cross-linking revealed TFE binding to a preformed open transcription bubble. In preinitiation complexes, the N-terminal part of TFE containing a winged helix-turn-helix motif is cross-linked specifically to DNA of the nontemplate DNA strand at positions –9 and –11. In complexes stalled at +20, TFE cross-linked specifically to positions +9, +11, and +16 of the non-template strand. Analyses of transcription complexes stalled at position +20 revealed a TFE-dependent increase of the resumption efficiency of stalled RNA polymerase and a TFE-induced enhanced permanganate sensitivity of thymine residues in the transcription bubble. These results demonstrate the presence of TFE in early elongation complexes and suggest a role of TFE in stabilization of the transcription bubble during elongation.

The orientation of DNA in an archaeal transcription initiation complex

RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase–promoter interface. The largest component of this archaeal polymerase is split into two subunits, A′ and A″, whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.

Archaeal transcription: Function of an alternative transcription Factor B from Pyrococcus furiosus.

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.

Rearrangement of the RNA polymerase subunit H and the lower jaw in archaeal elongation complexes

The lower jaws of archaeal RNA polymerase and eukaryotic RNA polymerase II include orthologous subunits H and Rpb5, respectively. The tertiary structure of H is very similar to the structure of the C-terminal domain of Rpb5, and both subunits are proximal to downstream DNA in pre-initiation complexes. Analyses of reconstituted euryarchaeal polymerase lacking subunit H revealed that H is important for open complex formation and initial transcription. Eukaryotic Rpb5 rescues activity of the ΔH enzyme indicating a strong conservation of function for this subunit from archaea to eukaryotes. Photochemical cross-linking in elongation complexes revealed a striking structural rearrangement of RNA polymerase, bringing subunit H near the transcribed DNA strand one helical turn downstream of the active center, in contrast to the positioning observed in preinitiation complexes. The rearrangement of subunits H and A′′ suggest a major conformational change in the archaeal RNAP lower jaw upon formation of the elongation complex.

Engines of gene expression

A backbone model of ten subunits of yeast RNA polymerase II has been derived from the ongoing analysis of its crystal structure. Notable features include ‘jaws’ for holding DNA, a putatively RNA-regulated ‘sliding clamp’, two ‘pores’ located in the vicinity of the catalytic center, and a high degree of similarity with the structure of a bacterial RNA polymerase.

Displacement of the transcription factor B reader domain during transcription initiation

Abstract Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.