Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Aim:To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryoticMSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme.Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed.Place and Duration of Study: School of Biotechnology, MaduraiKamaraj University, Madurai, India, between 2007-2013. Methodology:Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results:Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the templatebinding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9thposition from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regionsare highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, inthe eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6thposition are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes.Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present

Prokaryotic expression and application of RNA polymerase of SARS coronavirus / 解放军医学杂志

Objective To study the antigenicity of RNA polymerase and the relationship between the protein and the SARS-CoV replication. Methods The fragment was amplified by PCR, ligated with the prokaryotic expression vector pET32a+ and transformed into E. coli BL-21 .The expressed fusion protein identified by Western-blot and ELISA was used to detect the anti-SARS CoV IgG in different sera. Results The fusion protein was expressed successfully in E. coli BL-21. Detected by ELISA, the positive percentage of the anti-SARS IgG in the healthy donors, patients, infected animals and the rhesus administrated with the inactivated-virus were 0%, 95%, 100% and 0%, respectively. Conclusion Good antigenicity was shown in the expressed RNA polymerase. It can be used to diagnose the infection and to demonstrate the replication of SARS-CoV virus.

Effects of Whole Body Gamma Ray Irradiation on Transcrip-tional Activities of Nuclei of Rat Liver and Spleen / 第三军医大学学报

The transcriptional activities of the nuclei of the liver and spleen cells of rats were studied at the 1st, 4th, 18th, and 24th hour after whole-body irradiation with gamma rays. It was found that the transcriptional activity of the nuclei of rat spleen was invariably markedly suppresed in all the periods after irradiation while that of the rat liver demonstrated a diphasic change, that is, an exaggeration of the activity at the 4 th hour but a reduction at the 18th and 24th hour after irradiaiton. The change of the activity of RNA polymerase Ⅱ showed no difference from that of RNA polymerases Ⅰ+Ⅲ. The mechanism of these changes was briefely discussed.

Determination of transcription activity of rat liver nucleic free RNA Polymerase in vitro / 第三军医大学学报

The inhibitory effects of actinomycin D on chromatin (or DNA) template and the stimulating effects of synthetic template Poly [d(A-T)] on transcription were observed in the liver nucleic transcriptional system of the rat in vitor.By introducing proper concentrations of actinomycin D and Poly [d(A-T)] into each reactionary system,a method to assay free RNA polymerase activity in the nucleus was established.In addition,a comparison of the contents between chromatin-engaged and free polymerase in normal rat liver nuclei isolated with hypertonic sucrose (2.3 mol/L) was made.Eventually,the possible role free RNA Polymerases play in the eukaryotic transcriptional process was discussed.

Uterine RNA synthesis during protein deficiency and steroid-maintained pregnancy in rats.

Some aspects of uterine RNA synthesis including [3H]-uridine incorpo ration into RNA, activities of RNA polymerases and ribonucleases were studied. It was observed that both normal and pregnant animals, kept on protein-free diet for 15 and 20 days, showed a significant increase in in vivo uptake of [3H]-uridine into total RNA. Activities of RNA polymerase I and polymerase III increased two-fold in animals kept on a protein-free diet; however, RNA polymerase II activity was unaffected by protein restriction. In animals kept on protein-free diet where pregnancy was maintained by exogenous estrogen and progesterone, specific activity of nuclear RNA was further increased and the activities of RNA polymerases I, II and III markedly increased. Levels of RNase were also increased significantly during protein deficiency, thus showing a rapid turn-over of uterine RNA. These observations indicate that during protein restriction, uterine RNA synthesis is regulated at transcriptional level by a selective stimulation of RNA polymerase and RNase also plays an important role.