Talk abstracts

Talk on Friday 02:30-02:45pm submitted by Natalie Merlino

TRAP-mediated Transcription Termination at a Deficient Intrinsic Terminator

Natalie Merlino (Department of Biological Sciences, University at Buffalo), Paul Gollnick (University at Buffalo)

Abstract:
In the trpEDCFBA operon of Bacillus subtilis, transcription is regulated by the trp RNA-binding attenuation protein (TRAP) using an attenuation mechanism involving two competing RNA secondary structures. These mutually exclusive RNA structures, an antiterminator and terminator, are in the 203-nt 5’ leader region of the transcript upstream of trpE. When tryptophan is present the TRAP protein is activated and binds to a region in the trp leader RNA that contains 11 (G/U)AG repeats. This binding prevents formation of the antiterminator and allows formation of a terminator that induces transcription termination and the genes in the operon are not expressed. When tryptophan is limiting, TRAP does not bind to the RNA and an alternative antiterminator structure forms allowing expression of the trp genes. Mutations in the trp leader that prevent formation of the anti-terminator and allow the terminator structure to form constitutively fail to terminate in the leader region in the absence of TRAP in vitro. In the presence of TRAP, termination occurs suggesting that TRAP may have an additional role in attenuation other than changing the secondary structure of RNA. The characteristics of these mutants were also examined in vivo. Since the trp terminator does not function as an intrinsic terminator in the absence of TRAP, we wanted to determine the importance of the RNA structure/sequence in TRAP-mediated transcription termination. To do so mutations were made in the hairpin stem and the U-stretch of the trp terminator and transcription termination was examined in vitro and in vivo. While TRAP was able to cause termination at these mutant terminators in vivo, it was not able to do so in vitro. Therefore, there may be an additional factor present in vivo that allows TRAP to cause transcription termination at a deficient terminator. A genetic screen using transposon mutagenesis was performed and found a possible additional transcription factor, yceG.

Keywords: TRAP