| dc.description.abstract |
<p>Transcription is the process by which cells translate genetic information stored
in DNA into RNA. Transcription is a highly regulated and discontinuous process, and
elongation is frequently blocked by DNA damage, pause sites, or intrinsic or external
inhibitors. Due to the essential nature of transcription, the cell has numerous ways
of dealing with these blockages to transcription, only some of which are understood.
We examined the fate of RNA polymerase stalled by DNA-protein crosslinks as well as
elongation inhibitors Streptolydigin and Actinomycin D.</p><p> We use 5-azacytidine,
a cytosine analog that covalently traps cytosine methyltransferases, as a model system
for DNA-protein crosslinks (DPCs) in<italic>Escherichia coli</italic>. Our lab previously
showed the importance of the tmRNA system for survival during DPC-formation, implying
that transcription and translation are blocked by DPCs. For tmRNA to function, the
A-site must be cleared, requiring either RNA polymerase to be released first or the
nascent RNA chain to be cleaved. Using cell growth assays, we tested mutants related
to A-site cleavage factors known to affect transcription initiation, elongation, and
termination. Of these mutants, only DksA seemed to have a mild effect, and only at
late stages of growth phase. However, western blots for tmRNA tagging showed that
<italic>dksA</italic> mutants have increased rather than decreased tmRNA tagging,
indicating that another unknown factor is responsible for enabling tmRNA activity.
</p><p> Since the issue of repair of DPCs remains unresolved, and the repair of
DPCs could affect the blocked elongation complex, we used the same cell growth assay
to look for potential repair pathways. We found that <italic>dnaK</italic> knockouts
were slightly resistant to 5-azacytidine treatment which, coupled with our previous
finding that <italic>dnaJ</italic> mutants are hypersensitive to DPCs, implies a potential
DnaK-independent role for DnaJ in DPC repair.</p><p> Previous <italic>in vitro</italic>
studies have shown that Stl-stalled RNAP is stable, while <italic>in vivo</italic>
studies argued that Stl-inhibited polymerases are released from the DNA transcript,
implying that there is a release factor responsible for removing RNAP from DNA <italic>in
vivo</italic>. Using cell growth assays, Western blots for tmRNA tagging, and <italic>in
vitro</italic> studies, we showed the transcription-coupled repair factor Mfd is responsible
for releasing Stl-stalled RNAP, and that treatment with an elongation inhibitor such
as Stl is an effective treatment against cells overexpressing the transcription-coupled
repair pathway. </p><p> The tmRNA western blots also implied that Mfd has termination
abilities in wildtype cells, leading us to perform RNAseq analysis on <italic>mfd</italic>
knockout and overexpressing cells. We found that global transcription patterns are
changed by altering Mfd levels, thus allowing us to propose a novel transcription
regulatory role for Mfd.</p><p> We extended our elongation inhibitor studies
to the eukaryotic inhibitor Actinomycin D and found that transcription-coupled repair
pathway is again involved in responding to stalled RNAP. We also screened rifampicin-resistant
RNAP mutants for Actinomycin D resistance and found several with the desired phenotype.
We thus propose that Actinomycin D inhibition is more complicated than just steric
hindrance due to DNA intercalation.</p>
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