Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators
Jeng, Shih-Tong
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https://hdl.handle.net/2142/20724
Description
Title
Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators
Author(s)
Jeng, Shih-Tong
Issue Date
1991
Doctoral Committee Chair(s)
Gumport, Richard I.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Language
eng
Abstract
To investigate the mechanism of transcription termination by bacteriophage RNA polymerases, templates encoding variants of the transcription-termination structure in the regulatory region of the threonine (thr) operon of Escherichia coli were used. The thr terminator comprises two distinct structural elements: a G+C-rich inverted repeat, which encodes an RNA hairpin structure, and A+T-rich regions, one of which contains a continuous sequence of template deoxyadenosine residues within which the transcript terminates. Fourteen terminator variants were analyzed by T7 RNA polymerase and we find that not only the hairpin structure itself but also its sequence influences termination. Furthermore, the formation of a hairpin in the RNA encoded by the A+T-rich regions of the attenuator is not mandatory for termination.
Similar studies were also applied to T3 and Sp6 RNA polymerases. T3 RNA polymerase has very similar termination properties as T7 RNA polymerase does with both wild-type and mutated thr terminators. Sp6 RNA polymerase shows 63% termination efficiency compared to the 43% termination efficiency of T7 RNA polymerase on thr terminator, and the thr variants have less effect on Sp6 RNA polymerase than on T7 RNA polymerase.
A series of seven deletion variants that successively shorten the deoxyadenosine tract in the terminator template were also analyzed. Results from these experiments with T7 and Sp6 RNA polymerases indicate that complete readthrough occurs when there are four or fewer deoxyadenosine residues. With 5 template deoxyadenosine residues there is 5 percent termination increasing to 32 percent with 8 deoxyadenosines--the value produced by the wild-type terminator. In addition, a comparison with E. coli RNA polymerase shows that bacteriophage RNA polymerases require a more perfect region of dyad symmetry and a longer deoxyadenosine tract than does the bacterial enzyme to terminate with maximum efficiency.
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