Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...

The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5'-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping, and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

SummaryThe general subunit of all three eukaryotic RNA polymerases, Rpb12, and subunit P of the archaeal enzyme show sequence similarities in their N-terminal zinc ribbon and some highly conserved residues in the C-terminus. We report here that archaeal subunit P under the control of a strong yeast promoter could complement the lethal phenotype of a RPB12 deletion mutant and that subunit Rpb12 from yeast can functionally replace subunit P during reconstitution of the archaeal RNA polymerase. The DP enzyme is unable to form stable open complexes, but can efficiently extend a dinucleotide on a premelted template or RNA on an elongation scaffold. This suggests that subunit P is directly or indirectly involved in promoter opening. The activity of the DP enzyme can be rescued by the addition of Rpb12 or subunit P to transcription reactions. Mutation of cysteine residues in the zinc ribbon impair the activity of the enzyme in several assays and this mutated form of P is rapidly replaced by wild-type P in transcription reactions. The conserved zinc ribbon in the N-terminus seems to be important for proper interaction of the complete subunit with other RNA polymerase subunits and a 17-amino-acid C-terminal peptide is sufficient to support all basic RNA polymerase functions in vitro.