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

Main Article Content

Peramachi Palanivelu

Abstract

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 eukaryotic MSU 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, Madurai Kamaraj 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 template binding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9th position 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 regions are 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, in the 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 -6th position 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.

Keywords:
Multi-subunit DNA dependent RNA polymerases, eukaryotic RNA polymerases, RNA polymerase II, Saccharomyces cerevisiae, conserved motifs, polymerase active site, polymerization mechanism, transcription slippage diseases, drug design

Article Details

How to Cite
Palanivelu, P. (2019). Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism. International Journal of Biochemistry Research & Review, 26(3), 1-60. https://doi.org/10.9734/ijbcrr/2019/v26i330097
Section
Original Research Article

References

Anikin M, Molodtsov V, Temiakov D, McAllister WT. Transcript slippage and recoding. In: Atkins JF, Gesteland RF, Bujnicki JM. (eds). Recoding: Expansion of decoding rules enriches gene expression. 24th edn. Springer, New York. 2010;409–432.

Sahin U, Kariko K, Tǖreci Ö. mRNA-based therapeutics — Developing a new class of drugs. Nat Rev Drug Discov. 2014;13:759–780.

Conry RM, LoBuglio AF, Wright M, Sumerel L, Pike MJ. Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res. 1995;55:1397–1400.

Palanivelu P. Multi-subunit RNA Poly-merases of Bacteria - An insight into their active sites and catalytic mechanism. Indian J Sci Technol. 2018;11:1-37.

Roeder RG, Rutter, WJ. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature. 1969;224: 234–237.

Werner F, Grohmann D. Evolution of multisubunit RNA polymerases in the three domains of life. Nat Rev Microbiol. 2011;9: 85–98.

Domecq C, Trinhl V, Langelier MF, Archambault J, Coulombe B. Inhibitors of multisubunit RNA polymerases as tools to study transcriptional mechanisms in prokaryotes and eukaryotes. Curr Chem Biol. 2008;2:20–31.

Ma C, Yang X, Lewis PJ. Bacterial transcription as a target for antibacterial drug development. Microbiol Mol Biol Rev. 2016;80:139–60.

Ream TS, Haag JR, Pikaard, CS. Plant multisubunit RNA polymerases IV and V: in Murakami, KS, Trakselis, MA (eds.), Nucleic Acid Polymerases, Nucleic Acids and Molecular Biology. Springer-Verlag Berlin Heidelberg. 2014;30.

DOI: 10.1007/978-3-642-39796-7_13

Lane WJ, Darst SA. Molecular evolution of multisubunit RNA polymerases: Sequence analysis. J Mol Biol. 2010;395:671–85.

Sweetser D, Nonet M, Young RA. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. Proc Natl Acad Sci. USA. 1987; 84:1192–1196.

Minakhin L, Bhagat S, Brunning A, Campbell EA, Darst SA, Ebright RH, Severinov K. Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly. Proc Natl Acad Sci USA. 2001;98:892-897.

Nonet M, Sweetser D, Young RA. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987;50:909-915.

Todone F, Weinzierl R, Brick P, Onesti S. Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target, Proc Natl Acad Sci. USA. 2000;97:6306-6310.

Bushnell DA, Kornberg RD. Complete, 12-subunit RNA polymerase II at 4.1-Å resolution: Implications for the initiation of transcription. Proc Natl Acad. Sci. USA. 2003;100:6969–6973.

Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD. Structural basis of transcription: An RNA polymerase ii elongation complex at 3.3Å resolution; 2001.
Available:www.sciencexpress.org
DOI: 10.1126/science.1059495

Young RA. RNA Polymerase II. Ann Rev Biochem. 2003;60:689–715.

Hahn S. Structure and mechanism of the RNA polymerase II transcription machinery, Nat. Str. Biol. Mol. Biol. 2004; 11:394-403.

West ML, Corden JL. Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations, Genetics. 1995;140:1223-1233.

Sylvain E, Shona M. Cracking the RNA polymerase II CTD code. Trends Genet., 2008;24:280–288

Egloff S, O’Reilly D, Chapman RD, Taylor A, Tanzhaus K, Pitts L, Eick D, Murphy S. Serine 7 of the RNA polymerase II CTD is specifically required for snRNA gene expression. Science. 2007;318:1777–1779.

Phatnani HP, Greenleaf AL. Phosphoryla-tion and functions of the RNA polymerase II CTD. Genes Dev. 2006;20:2922-2936.

McCracken S, Fong N, Rosonina E, Yankulov K, Brothers G, Siderovski D, Hesse A Foster S, Shuman S, Bentley DL. 5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA poly-merase II. Genes Dev. 1997;11:3306-3318.

Fong N, Bentley DL. Capping, splicing, and 3′ processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD. Genes Dev. 2001;15:1783–1795.

Kolodziej PA, Woychik, N, Liao SM, Young RA. RNA polymerase II Subunit composi-tion, stoichiometry, and phosphorylation. Mol Cell Biol. 1990;10:1915-1920.

Sheffer A, Varon M, Choder M. Rpb7 can interact with RNA polymerase II and support transcription during some stresses independently of Rpb4. Mol. Cell. Biol. 1999;19:2672–2680.

Woychik NA, Young RA. Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA. 1992;89:3999-4003.

Cramer P, Bushnell DA, Kornberg RD. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science. 2001;292:1863-76.

Palanivelu P. Single subunit RNA Poly-merases – An insight into their active sites and mechanism of action, Biotech J Int. 2017;20:1-35.

Palanivelu P. Active sites of the multi-subunit RNA polymerases of Eubacteria and chloroplasts are very similar in Structure and Function. Indian J Sci Technol. 2019;12:1-32.

Cramer P. Multisubunit RNA polymerases. Curr Opin Struct Biol. 2002;12:89–97.

Pal M, Luse DS. The initiation–elongation transition: Lateral mobility of RNA in RNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23. EMBO J. 1997;16:7468–7480.

Fiedler U, Timmers HTM. Analysiis of the open region of RNA polymerase II transcription complexes in the early phase of elongation. Nucleic Acids Res. 2001;29: 2706-2714.

Palanivelu P. DNA polymerases – An insight into their active sites and mechanism of action, Int. J. Biochem. Res. Rev. 2013;3:205-247.

Tunitskaya VL, Kochetkov SN. Structural and functional analysis of bacteriophage T7 RNA polymerase. Biochemistry (Moscow). 2002;67:1124–35.

Zhang G, Campbell EA, Minakhin L, Richter C, Severinov K, Darst SA. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Cell. 1999; 98:811-824.

Kostyuk SM, Dragan DL, Lyakhov VO, Rechinsky VL, Tunitskaya BK. Chernov SN, Kochetkov E. Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA. FEBS Lett. 1995; 369:165–168.

Hausmann S, Shuman S. Characterization of the CTD Phosphatase Fcp1 from Fission Yeast: Preferential dephosphoryla-tion of serine 2 versus serine 5. J Biol Chem. 2002;277:21213-21220.

Svetlov V, Vassylyev DG, Artsimovitch I. Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase. J Biol Chem. 2004;279:38087-90.

Trinh V, Langelier MF, Archambault J, Coulombe B. Structural perspective on mutations affecting the function of multisubunit RNA polymerases. Microbiol Mol Biol Rev. 2006;70:12–36.

Kaplan CD, Larsson KM, Kornberg RD. The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by alpha-amanitin. Mol Cell. 2008;30:547–556.

Wang D, Bushnell D, Westover K, Kaplan C, Kornberg RD. Structural basis of transcription: Role of the trigger loop in substrate specificity and catalysis. Cell. 2006;127:941–954.

Campbell EA, Korzheva N, Mustaev A, Murakami K, Nair S, Goldfarb A, Darst SA. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell. 2001;104:901–912.

Kireeva ML, Komissarova N, Waugh DS, Kashlev M. The 8-nucleotide-long RNA: DNA hybrid is a primary stability determinant of the RNAP II elongation complex. J Biol Chem. 2000;275:6530–6536.

Zaychikov E, Denissova L, Meier T, Gotte M, Heumann H. Influence of Mg2+ and temperature on formation of the transcript-tion bubble. J Biol Chem. 1997;272:2259–67.

Luse DS. Promoter clearance by RNA polymerase II. Biochim Biophys Acta. 2013;1829:63–68.

Pal M, Ponticelli AS, Luse DS. The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell. 2005;19:101-110.

Giardina C, Lis JT. DNA melting on yeast RNA polymerase II promoters. Science. 1993;261:759-762.

Holstege FCP, Fiedler U, Timmers HTM. Three transitions in the RNA polymerase II transcription complex during initiation. EMBOJ. 1997;16:7468–7480.

Barnes CO, Calero M, Malik I, Graham BW, Spahr H, Lin G, Cohens A, et al. Crystal structure of a transcribing RNA polymerase II complex reveals a complete transcription bubble. Mol Cell. 2015;59: 258-269.

Severinov K, Mustaev A, Kukarin A, Muzzin O, Bass I, Darst SA, Goldfarb A. Structural modules of the large subunits of RNA polymerase. Introducing archae-bacterial and chloroplast split sites in the beta and beta’subunits of Escherichia coli RNA polymerase. J Biol Chem. 1996;271: 27969–27974.

Sydov JH, Cramer P. RNA polymerase fidelity and transcriptional proofreading. Curr Opin Struct Biol. 2009;19:732-9.

Nudler E. RNA polymerase active center: The molecular engine of transcription. Ann Rev Biochem. 2009;78:335–361.

Sosunov V, Sosunova E, Mustaev A, Bass I, Nikiforov V, Goldfarb A. Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase. EMBO J. 2003;22:2234–44.

Liu X, Bushnell DA, Kornberg RD. RNA polymerase II transcription: Structure and mechanism. Biochim Biophys Acta. 2013; 1829:2-8.