Functional analysis of Methanocaldococcus jannaschii RNA polymerase A’ ‘bridge-helix’ using a fully automated high-throughput robotic system
Author(s)
Tan, Lin
Type
Thesis
Abstract
RNA polymerases (RNAPs) are the ‘engines’ of cellular transcriptional machineries,
which are essential to life and highly conserved from bacteria to eukaryotes. Though
crystal structures of both eukaryotic and bacterial RNAPs have been intensively
studied, the relationships between structures and appropriate functions of such
enzymes in eukaryotes remain unknown since there has not yet been possible to
constitute any active eukaryotic RNAPs from recombinant subunits. The successfully
assembled archaeal counterparts have provided an alternative approach to study the
eukaryotic system due to not only the structural similarities between these enzymes
but also the structural and functional similarities of their basal transcriptional
machineries.
‘Bridge-helix’ is one of the most highly conserved structures near the catalytic site of
RNAPs, which has been proposed to play an important role in coordinating the
processing of nucleic acid substrates through the active center. 17 adjacent residues
(mjA’-L814 to mjA’-R830) within the central portion of Methanocaldococcus
jannaschii A’ ‘bridge-helix’ were chosen for a systematic high-throughput sitedirected
mutagenesis approach using a novel robotic system. This robotic system is
fully automated without any human interventions that may enormously reduce human
errors and effectively increase the number of samples that could be processed in
parallel.
The results obtained from such high-throughput approach showed a wide spectrum of
in vitro phenotypes ranging from complete loss of function to ‘superactivity’.
According to the unexpected functional evidences obtained with the ‘superactive’
mutants, we propose a highly favorable kinked ‘bridge-helix’ conformation for the
nucleotide addition cycle that has to be precisely localized in certain positions in order
to increase the specific activity of RNAPs. The fact that no additive effects have been
found so far in any of the ‘superactive’ double mutants suggests that various single
amino-acid substitution ‘superactive’ mutants may affect the same process in a
functionally overlapping and mutually independent manner.
which are essential to life and highly conserved from bacteria to eukaryotes. Though
crystal structures of both eukaryotic and bacterial RNAPs have been intensively
studied, the relationships between structures and appropriate functions of such
enzymes in eukaryotes remain unknown since there has not yet been possible to
constitute any active eukaryotic RNAPs from recombinant subunits. The successfully
assembled archaeal counterparts have provided an alternative approach to study the
eukaryotic system due to not only the structural similarities between these enzymes
but also the structural and functional similarities of their basal transcriptional
machineries.
‘Bridge-helix’ is one of the most highly conserved structures near the catalytic site of
RNAPs, which has been proposed to play an important role in coordinating the
processing of nucleic acid substrates through the active center. 17 adjacent residues
(mjA’-L814 to mjA’-R830) within the central portion of Methanocaldococcus
jannaschii A’ ‘bridge-helix’ were chosen for a systematic high-throughput sitedirected
mutagenesis approach using a novel robotic system. This robotic system is
fully automated without any human interventions that may enormously reduce human
errors and effectively increase the number of samples that could be processed in
parallel.
The results obtained from such high-throughput approach showed a wide spectrum of
in vitro phenotypes ranging from complete loss of function to ‘superactivity’.
According to the unexpected functional evidences obtained with the ‘superactive’
mutants, we propose a highly favorable kinked ‘bridge-helix’ conformation for the
nucleotide addition cycle that has to be precisely localized in certain positions in order
to increase the specific activity of RNAPs. The fact that no additive effects have been
found so far in any of the ‘superactive’ double mutants suggests that various single
amino-acid substitution ‘superactive’ mutants may affect the same process in a
functionally overlapping and mutually independent manner.
Date Issued
2009-03
Date Awarded
2009-03
Advisor
Weinzierl, Robert
Creator
Tan, Lin
Publisher Department
Department of Life Sciences, Division of Cell and Molecular Biology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)