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Research Article Open access CC BY 4.0

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

Peramachi Palanivelu

International Journal of Biochemistry Research & Review · pp. 1–60 · Published 17 Jul 2019

10.9734/ijbcrr/2019/v26i330097

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.

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

References (29)

  1. 1 Structural Mechanism for Rifampicin Inhibition of Bacterial RNA Polymerase [DOI]
  2. 2 Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution [DOI]
  3. 3 Multiple Forms of DNA-dependent RNA Polymerase in Eukaryotic Organisms [DOI]
  4. 4 Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution [DOI]
  5. 5 Structure and mechanism of the RNA polymerase II transcription machinery [DOI]
  6. 6 Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis [DOI]
  7. 7 Evolution of multisubunit RNA polymerases in the three domains of life [DOI]
  8. 8 Cracking the RNA polymerase II CTD code [DOI]
  9. 9 Complete, 12-subunit RNA polymerase II at 4.1-Å resolution: Implications for the initiation of transcription [DOI]
  10. 10 The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin [DOI]
  11. 11 Construction and analysis of yeast RNA polymerase II CTD deletion and substitution mutations. [DOI]
  12. 12 Molecular Evolution of Multisubunit RNA Polymerases: Sequence Analysis [DOI]
  13. 13 DNA Melting on Yeast RNA Polymerase II Promoters [DOI]
  14. 14 The Role of the Transcription Bubble and TFIIB in Promoter Clearance by RNA Polymerase II [DOI]
  15. 15 Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble [DOI]
  16. 16 Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II [DOI]
  17. 17 Bacterial RNA polymerase subunit ω and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly [DOI]
  18. 18 Three transitions in the RNA polymerase II transcription complex during initiation [DOI]
  19. 19 Characterization of the CTD Phosphatase Fcp1 from Fission Yeast [DOI]
  20. 20 Discrimination against Deoxyribonucleotide Substrates by Bacterial RNA Polymerase [DOI]
  21. 21 Structural Modules of the Large Subunits of RNA Polymerase [DOI]
  22. 22 Rpb7 Can Interact with RNA Polymerase II and Support Transcription during Some Stresses Independently of Rpb4 [DOI]
  23. 23 Influence of Mg2+ and Temperature on Formation of the Transcription Bubble [DOI]
  24. 24 The initiation–elongation transition: Lateral mobility of RNA in RNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23 [DOI]
  25. 25 Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae. [DOI]
  26. 26 Plant Multisubunit RNA Polymerases IV and V [DOI]
  27. 27 "Multi-subunit RNA Polymerases of Bacteria: An insight into their Active Sites and Catalytic Mechanism" [DOI]
  28. 28 Analysis of the open region of RNA polymerase II transcription complexes in the early phase of elongation [DOI]
  29. 29 5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II [DOI]

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