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|Title:||Transcriptional regulation of the Bacillus subtilis pyrimidine biosynthetic operon|
|Doctoral Committee Chair(s):||Switzer, Robert L.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5$\sp\prime$ to 3$\sp\prime$ order: a 150 nt untranslated leader; pyrR, which encodes a 21 kDa protein; a 173 nt intercistronic region; pyrP, which encodes a 46 kDa protein; a 145 nt intercistronic region; followed by 8 overlapping cistrons encoding all of the 6 enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via a PryR-mediated attenuation mechanism at 3 attenuators within the operon, which are located in the 5$\sp\prime$ leader, the pyrP-pyrP intercistronic, and the pyrP-pyrB intercistronic regions. Each of the attenuators is predicted to be comprised of an antiterminator (AT) stem-loop structure, a terminator (T) stem-loop structure which overlaps with the 3$\sp\prime$ strand of the AT, and a consensus sequence which is proposed to be the binding site for the regulatory protein PyrR, and is also capable of forming an anti-antiterminator (AAT) stem-loop structure that competes with the 5$\sp\prime$ strand of the AT.
pyr-lacZ fusion experiments showed that the nutritional regulation of each attenuator separately was roughly equivalent and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR. The participation of Spo0A was excluded. A series of titration plasmids was used to demonstrate that the regulatory protein PyrR is an RNA-binding attenuation protein.
An in vitro transcription system was used to study the transcriptional regulation of the pyrimidine biosynthetic operon from B. subtilis. The three DNA templates used in this system specified the three attenuation regions in the B. subtilis pyr operon. Transcription attenuation in all three templates was dependent on the PyrR regulatory protein and a coeffector, UMP or UTP at higher concentration. PRPP, a substrate for uracil phosphoribosyl transferase reaction which PyrR is able to catalyze, antagonized the UMP effect on PyrR-mediated attenuation.
A series of antisense deoxyoligonucleotides was used to disrupt the potential stem-loop structures in the attenuation regions. The abilities of these deoxyoligonucleotides to affect termination in the in vitro transcription system in the predicted manner strongly supported the existence of the AAT, AT, and T stem-loop structures and their function in regulation of the pyr operon.
A modified model for transcriptional regulation of the B. subtilis pyr operon is proposed, in which each attenuator region specifies two sets of alternative stem-loop structures: AT versus AAT+T. At low concentrations of UMP, PyrR is unable to bind to pyr RNA, and the AT structure is favored to form, resulting in readthrough transcription. At high concentrations of UMP, the binding of the activated PyrR to the AAT structure stabilizes the AAT+T structure at the expense of the AT structure, resulting in transcription termination.
|Rights Information:||Copyright 1996 Lu, Yang|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9702590|