Biological systems under study
Single molecule studies of transcription
RNA polymerase is the enzyme which is responsible for transcribing the
sequence of a double stranded DNA molecule into a single stranded
RNA molecule. In bacteria,
such as E. coli., the resulting messenger RNA is fed
directly into a ribosome, which constructs a protein
with the specified sequence of amino acids.
In Eukaryotes
the process is considerably more complex, since the DNA must
be extracted from the highly compact form it takes in a
chromosome before it can be transcribed, and the resulting RNA
undergoes considerable post-processing before it is ready
to be translated into a protein.
However, the catalytic core of the Prokaryotic and Eukaryotic
varients of RNA polymerase are highly conserved and it is clear
that they are quite similar in their basic mode of operation.
RNA Polymerase is a processive enzyme that crawls down the DNA molecule,
synthesizing an RNA strand that is templated against one strand
of the DNA molecule. Schematically, it is configured as shown below.
The basic features of the schematic are reflected
in the crystal structure of the prokaryotic enzyme. This is
evident in the following image, based on the structures from
Seth Darst's lab and rendered by Elio Abbandonzieri.
The protein (shown in green) contains channels where the
two strands of the DNA double helix are separated. This exposes the
bases of the DNA so that
a DNA-RNA hybrid can form. In this way the DNA serves as
a template against which the RNA polymer is extended by ligation
of corresponding nucleotides onto the nascent RNA polymer.
Another interesting aspect of RNA
polymerase is that the energy released by the breaking of a
phosphate bond in the incoming nucleotide tri-phosphate (NTP)
provides the energy that drives the enzyme forward.
Transcription has been extensively studied in bulk biochemical
assays, typically by collecting the RNA transcript created after
transcriptional elongation has been allowed to proceed for a
finite time in an initially synchronized population of
elongation complexes.
An great deal of information can be obtained from such assays.
In the gel shown above, which run by Karen Adelman and
assays transcription of a typical bacterial gene at
saturating nucleotide conditions, it is evident that
the elongation rate of RNA polymerase molecules
have a considerable spread. It is also clear that the molecules have a
tendency to pause at specific locations on the template. (Notice
the dark bands that appear in all lanes of the gel.)
However, the bulk assays do not allow us to determine how
the speeds of individual molecules vary and how different molecules
respond to the pause sites. For instance, does every molecule stop
at every pause site, or do some molecules pause at some sites
and not others?
RNA Polymerase is an ideal candidate for single molecule manipulation
study because its function is intimately related the the movement
of the molecule down the DNA template. It is possible, in principle,
to watch a gene transcribed base by base by monitoring the movement
of the enzyme down the template. The experiment was first done
using optical tweezers in a configuration similar to that shown
below.
Using this configuration traces of RNAP polymerase position
as a function of time can be generated, in which the molecule
is seen to move along the template, halt for a period of time,
then resume. Several such traces are shown in the plot that follows.
One thing which is evident after inspection of the plot is that
molecules that take a longer or shorter time to transcribe the
template do not differ in their velocity when they are active. The
traces mainly vary in the fraction of time that the molecules
spend in the paused state.
In prior work, I have been involved in studies of the detailed
statistics of pauses in wild type and mutant RNAP molecules [1],
and of the mechanism of transcriptional inhibition
by an anti-bacterial protein factor [2]. In more recent work I
have been involved in an investigation of the
sequence dependence of transcriptional pausing [3]. (Please refer
to the references available below for details.)
As I start up my lab here at Maryland I plan to continue to investigate
basic issues in transcription. Interesting issues are how
regulatory factors modify the kinetics of transcription, how torsional
stress on the DNA affects transcriptional initiation, elongation
and termination, and how RNA polymerase II is able to transcribe
DNA that is bound by histone proteins and other protein factors.
References
[1] Adelman, K., La Porta, A., Santangelo, T. J., Lis, J. T., Roberts, J. W., Wang, M. D., PNAS 99, p. 13538-13543 (2002)
[2] Adelman, K., Yuzenkova, J., La Porta, A., Zenkin, N., Lee, J., Lis, J. T., Borukhov, S., Wang, M. D., Severinov, K., Molecular Call 14 p. 753-762 (2004)
[3] Herbert, K. M., La Porta, A., Wong, B. J., Mooney, R. A., Neuman, K. C., Landick, R., Block, S. M., Cell 125 p. 1083-1094 (2006)