|
THE IRE RNA. |
Current
Hall Lab Projects
|
|||
|
|
Structure of the Mg2+-Riboswitch
(Dr. Rebecca L. Coppins) To characterize this novel
Riboswitch, its structure will be
mapped using enzymatic probes, hydroxyl radical footprinting, and terbium
cleavage. Properties of RNA mutants that have lost Mg2+ responsiveness
in vivo, and individual excised structural elements, will be compared to full
length wild type UTR RNA. Conditions that favor conformational transitions of
the UTR will be examined by varying the salt, temperature, and Mg2+
concentration in the solution. Different
conformations of the UTR RNA could be favored by the rate of transcription,
and could be controlled by the Mg2+ concentration. Because E. coli and Salmonella RNA polymerases are virtually identical, the E. coli enzyme will be used in
transcription assays to map the pause sites and the extent of termination in
wild type and mutant UTR RNAs, under conditions of varying Mg2+
concentration. We predict that the RNA will adopt alternative conformations
during specific polymerase pauses, and that the co-transcriptional folding of
the riboswitch will determine its functional form. To
correlate regulatory capability of the mgtA UTR with transcription patterns,
we will repeat these in vitro kinetics experiments using mutant UTRs. Patterns of transcription of mgtA UTR
mutants that are known to have lost regulatory ability (e.g. Loop A GGAGA to
CCUAU substitution; Stem A 5’ deletion), or those that fail to show any
activity in vivo (e.g. Stemloop A deletion), will be compared to the patterns
from wild-type UTR RNA. We predict that sites of termination will be absent
from transcripts of constitutively active mutants, and that a terminated
transcript (C220 or G178?) will be the only product of inactive mutants.
Through a comparison of these data, a map of predicted RNA folds and
resulting transcription reactions will be constructed, which will be tested
by additional site-directed mutations of the UTR.
E.
coli RNA polymerase transcription kinetics. Experiments at 37° C, with
100 μM ATP, 25 μM CTP and UTP for initiation, then addition of 25 μM GTP
for elongation (32P-CTP for observation). MgCl2
concentration is 10 mM. Times of assays are in minutes. The last assay is
taken after addition of all NTPs to a final concentration of 1 mM.
|
|||
Return to Washington University Biochemistry and Molecular Biophysics Homepage
Dr. Kathleen B. Hall (hall(at)biochem.wustl.edu)
Department of Biochemistry and
660
office: 314-362-4196
lab: 314-362-4197
or 314-747-8079
FAX: 314-362-7183
send email to kathleenhal(at)gmail.com
Last Update:
This site maintained by Tom Stump
Send site related comments or concerns to tstump(at)wanda.wustl.edu