Mechanisms of Transcription Elongation and
Its Regulation In Bacteria and Yeast
Transcription, the central step in gene
expression and regulation, is carried out by DNA-dependent RNA
polymerase (RNAP). Cellular RNAPs are large, multisubunit
assemblies. Their complexity presumably reflects an involvement
in interactions with numerous regulatory signals and factors that
modulate the activity at all stages of transcription. Recent
progress in the understanding of transcriptional mechanisms was
essentially attributed to the studies on the primary stage of
transcription cycle (promoter binding and activation). Although
the following steps of transcription (elongation and termination)
were shown to be the major target for gene regulation as well,
our knowledge about their mechanisms is considerably less
compelling.
Our research is focused on structural
understanding of the mechanisms of transcription elongation and
its regulation in E.coli and yeast systems. To this end we
undertook biochemical and structural characterization of the
basic intermediates of elongation - the ternary complexes that
composed of elongating form of RNAP, DNA template and RNA
product. Using the immobilized transcription system - an approach
that allows one to generate defined elongation complexes stalled
at desired positions on DNA, in combination with footprinting,
affinity crosslinking, and protein chemistry methods we have
build the new structure-functional model of the elongation
complex (see the figure below). This model provide a key to the
mechanism of transcription processivity and proofreading. It also
has far reaching implications for understanding of regulatory
mechanisms of elongation such as termination and pausing. Using
this model as a starting point we now pursue two main projects:
1) Structural analysis of transcription
elongation complexes from E.coli and yeast. Combining the
approaches mentioned above together with an electron
microscopy we propose to generate a detailed 3 dimensional
topological map of ternary elongation complexes based on the
available low resolution structure of E.coli and yeast RNAP
II. On this map certain protein regions within RNAP subunits
will be assigned to the principal functional domains.
2) Studies on molecular mechanisms that regulate
transcription elongation. The thrust of this project is to
determine the key intermediates in the pathways leading to
termination, pausing or arrest of the elongation by E.coli
RNAP and yeast RNAP II and how the elongation factors can
modulate these pathways.
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