The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation

Summary TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation.

Rpo2''-Q373 (red) are shown together with the X-ray structure of the archaeal polymerase of S. solfataricus (pdb file: 2pmz) (Hirata et al., 2008). This figure was prepared using PyMOL.

Ribbon Domain (E-H) of TFE
Shown are the frame-wise histograms of (A) TFE G44 Table S1 and S2).

Figure S4. Structure of the TBP-TFB core -SSVT6 Promoter Complex
In order to measure fluorescence quenching between TBP and the nontemplate strand (NTS) at position -21 we assembled preinitiation complexes with fluorescently labelled TBP (S72C-Alexa555, highlighted with red spheres), TFB, TFE, RNAP and a SSV T6 promoter oligonucleotides containing the black hole quencher BHQ-2 at position -21 (highlighted with red spheres). The distance between TBP S72 and NTS T(-21) in in the TBP/TFB core -TATA complex is 35.3 Å (TATA consensus sequence is highlighted in red).
The structure in the figure is Pyrococcus furiosus TBP (blue) and the TFB core domain (green, encompassing the N-and C-terminal cyclin repeats) bound to the viral SSV T6 promoter, which is used throughout this study (Littlefield et al., 1999). The template includes a short heteroduplex region (-3/+1) that stabilised the PIC by forming the open complex. The TATA box is highlighted in red. position 257 (small green mesh). The sizes of the density surfaces and meshes corresponds to 68% credible volumes. For comparison the eukaryotic TFIIE X-linking sites on the RNAPII are highlighted as red spheres. Hahn and co-workers derivatised the large RNAPII subunits RPB1 and RPB2 with the photoactivatable crosslinker Bpa, formed PICs and identified the polypeptides that were conjugated to specific incorporation sites (Chen et al., 2007). The eukaryotic TFIIE factor was cross linked to residues RPB1 His-213 and -286 highlighted as red spheres on the structure of the archaeal RNAP (corresponding to residues Rpo1 Lys-186 and Gln-259 in S. solfataricus RNAP, pdb 2PMZ).

Figure S7. Promoter Directed Transcription by Recombinant M. jannaschii RNAP Is Strictly Dependent on Basal Transcription Factors
Promoter directed transcription assays were carried out according to Experimental Procedures. The synthesis of the 70 nt runoff transcript is strictly dependent on the presence of the two transcription factors TBP and TFB. All values given in Å.

Production of Labeled TFE and RNAP Derivatives
Protein Production. Amber codons were engineered into genes encoding C- glycerol, 20 mM imidazole) and subsequently eluted in the same buffer containing 250 mM imidazole. Typical yields were 4 mg/l expression culture for TFE derivatives and 4-7 mg/l expression for RNAP-subunit derivatives Rpo1'257 and Rpo2"373. Rpo5-11C and Rpo7-49C and -65C derivatives respectively, were prepared as described in (Grohmann et al., 2009).
Protein Chemical Modification. TFE and RNAP-subunit derivatives containing site-specifically incorporated p-azido-L-phenylalanine were labeled by Staudinger ligation using phosphine derivatives of fluorescent probes (Chakraborty et al., 2010;Kiick et al., 2002). TFE derivatives were labeled using Cy3B-phosphine (Chakraborty et al., 2010); RNAP-subunit derivates were labeled with DL649-phosphine (Pierce). Following reaction of 0.5 mg TFE derivative or 6 mg of RNAP-subunit derivative with a 5-fold molar excess of fluorophore-phosphine for 2-16 h at 37°C, beta-mercaptoethanol was added to 1 mM, and, in the case of TFE derivatives, the product was isolated and refolded on a 1-ml Ni-NTA column (GE Healthcare; methods for isolation and refolding as in preceding section). Labelling efficiencies were ~50% for TFE derivatives and ~10-40% for RNAP-subunit derivatives. Rpo5 and Rpo7 derivatives were labeled as described in (Grohmann et al., 2009).
In vitro reconstitution of M.jannaschii RNAP including fluorescently labeled RNAP subunits. Fluorescently labeled RNAP subunits were directly introduced into RNAP reconstitution reactions following protocols described earlier (Werner and Weinzierl, 2002). The excess of non-coupled fluorophorephosphine was removed during the dialysis steps of the reconstitution process and additionally by size-fractionation the RNAP in a gel filtration run (Superose 6, GE Healthcare).

Nanopositioning System Experiments
Determination of the winged helix and the zinc-ribbon domain of TFE by using NPS The X-ray structure of the archaeal polymerase of S. solfataricus (Hirata et al., 2008) was used as a reference frame for the position calculation. Moreover, the volume occupied in the crystal structure was used as a restriction for the possible positions of the dye molecules. We assumed zero probability density within an already occupied volume, which was the volume of the protein shrunk by 1 Å to account for uncertainties in the x-ray structure, and equal probability density elsewhere in order to calculate the ADM prior.
The recently developed NPS method (software freely available at http://www.cup.uni-muenchen.de/pc/michaelis/) was applied separately for the two different positions of TFE using the measured FRET efficiencies and the information about the possible SDM positions and Förster distances as described above. As a result we obtain the probability density function for the winged helix and the zinc-ribbon domain of TFE. From this we calculate the smallest volumes that enclose a certain probability, so-called credible volumes. The surface of the credible volume was displayed by using the interactive visualisation program PyMOL.

Determination of the isotropic Förster radius
For each donor-acceptor pair the isotropic Förster radius R 0 iso was determined using standard procedures (Vamosi et al., 1996). First, the quantum yield of the donor sample was determined using Rhodamine 101 dissolved in ethanol as a standard (QY = 100 %). Second, overlap integrals were calculated from recorded donor emission spectra (528 to 700 nm with an excitation wavelength of 523 nm) and acceptor absorption spectra (400 to 700 nm). The winged helix domain of TFE was labelled at position G44APA with the donor dye Cy3B. The quantum yield of the donor was determined to 71 % and the calculated isotropic Förster radius R 0 iso resulted in 69 Å. The zinc-ribbon domain TFE-G133APA was labelled with the donor dye DyLight549. We assume a fluorescence quantum yield of 6 % (http://www.thermo.com/eThermo/CMA/PDFs/Various/File_9349.pdf) and calculated the isotropic Förster radius R 0 iso of 45 Å.
In order to account for uncertainties in the Förster distance due to orientation effects we then measured the steady state fluorescence anisotropies of the donor and acceptor dyes for all attachment sites (results shown in Table 3).
Assuming that there is no additional rotational movement beyond the time scale of the fluorescence lifetime, Monte Carlo Simulations were performed in order to calculate the probability densities of the Förster distances assuming an isotropic distribution of the average dye molecule orientation (Muschielok et al., 2008). For all donor-acceptor pairs the calculated probabilities were fitted using a sum of 10 Gaussian distributions and used in the NPS analysis as described previously (Muschielok et al., 2008).
Experimental setup for sp-FRET, data collection and analysis All sp-FRET experiments were performed on a homebuilt prism-based total internal reflection fluorescence microscope (TIRFM) described previously . Briefly, a frequency-doubled Nd:YAG laser (532 nm, Spectra-Physics) was used for the excitation of donor molecules and a He-Ne laser (637 nm, Coherent) for the direct excitation of the acceptor molecules.
The fluorescence signal of donor and acceptor was combined spatially by the use of a dichroic mirror. Fluorescence intensity was collected through a waterimmersion objective (Plan Apo 60X, NA 1,2, Nikon) and directed to an EMCCD camera (iXon DU-897E-CS0-BV, Andor). PIC complexes were immobilised onto the surface of a microfluidic chamber surface via PEG-Biotin/Neutravidin/Biotin as described previously . All measurements were recorded with an exposure time of 100 ms per frame for the duration time of 40 s. The acquired data was analysed using customwritten MATLAB software. We used a fully automated routine to find FRET pairs using an intensity threshold for the acceptor signal during the FRET measurement. The algorithm then calculates and subtracts the local background and computes fluorescence trajectories .
For the calculation of the FRET efficiency of each individual FRET pair, we used the following formula: for each FRET pair only data where acceptor photobleaching occurred prior to donor photobleaching were used in the data analysis .
As a result no zero FRET peak is visible in the histograms. The resulting histograms were computed either for every time point (frame-wise histogram) or using the determined mean FRET efficiencies of every molecule (moleculewise histogram). The histograms were then fitted with one or more Gaussian distributions and the mean FRET efficiency and its standard error were determined from the fit (see Table 1 and 2). These results were then used for further analysis with NPS (Muschielok et al., 2008).
Uncertainty in the position of dye molecules attached to known positions Satellite dye molecules (SDMs) were attached to known positions within the archaeal polymerase using flexible linkers. While the attachment point is known from the x-ray structure of the archaeal polymerase of S. solfataricus (pdb file: 2pmz) (Hirata et al., 2008), the precise location of the dye molecule is not. For the NPS analysis we therefore calculated the volume that is sterically accessible to the dye molecules, given the point of attachment, size of the dye molecule and the linker length (Muschielok et al., 2008). To this end, the SDMs were approximated by a sphere of diameter d dye and linked to the protein complexes by flexible linkers of dimensions L linker and d linker (see Table S3). We assume each SDM position within this accessible volume equally probable and for calculation purposes approximate the resulting probability density function by a sum of 10 Gaussian distributions. These Gaussians are used in the NPS analysis to describe the uncertainty of the SDM position (Muschielok et al., 2008) (see Figure S2).