TrfA initiator protein mutants altered for binding to plasmid RK2 origin

Document Type

Conference Presentation

Department

Biological Sciences

Conference Title

Keystone Symposium: Molecular Mechanisms in DNA Replication and Recombination

Location

Taos, NM

Conference Dates

January 25 - February 1, 1992

Date of Presentation

1-25-1992

Journal Publication

Journal of Cellular Biochemistry

ISSN

0730-2312

DOI

10.1002/jcb.240500602

Volume

50

Issue

S16B, Supplement: Keystone Symposia on Molecular & Cellular Biology

Publication Date

1992-01-25

First Page

F 360

Abstract

We have been studying interactions between the RK2 replication, initiation protein, TrfA, and the plasmid's origin sequences. An in vivo antibiotic selection system for isolating genes encoding sequence specific DNA binding proteins was adapted for use with TrfA. A binding site consisting of two 17-bp iterons separated by a 6-bp spacer region was cloned into the vector pNN388 (1) such that an increased level of spectinomycin resistance was dependent on TrfA specific biding to the iteron sequences. The results with the in vivo system were found to correlate well with observations of in vitro DNA binding activity of several previously characterized TrfA mutants. The system was then used to isolate mutants which were either defective or more effective in DNA binding. These latter mutants were found to fall into three classes: those active with only the complete (eight-iteron) RK2 origin, with only the minimal (five-iteron) RK2 origin, or with both types of origin. Additional properties of these different classes of mutant TrfA proteins will be described.

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