Date

1-4-2016 12:00 AM

Major

Microbiology

Department

Microbiology

College

College of Agriculture and Life Sciences

Project Advisor

Gregory Phillips

Project Advisor's Department

Veterinary Microbiology and Preventive Medicine

Description

The signal recognition particle (SRP) is a highly conserved molecular machine that targets hydrophobic polypeptides to cellular membranes of both eukaryotes and prokaryotes. Escherichia coli is an attractive system to study the system since the SRP is composed of a single protein (Ffh) and a small structural RNA (4.5S RNA). While much is known about the biochemical activity of the SRP in E. coli, how the cellular localization of Ffh and 4.5S RNA influences their activity is not as well understood. To approach this question, we have used green fluorescent protein (GFP) and the flavin-based fluorescent protein iLOV to tag the Ffh protein at both the amino- and carboxy-termini. In addition, we have also used the Broccoli fluorescent RNA to localize 4.5S RNA within living cells. Upon completion of tests to observe each construct’s ability to complement an ffh deletion mutation, we discovered that fluorescent tagging altered the function of the Ffh protein. In characterizing the gene fusions it was observed that GFP fusions to either the amino- or carboxy terminus resulted in a dominant negative phenotype. While the fluorescent tags did interfere with protein function, the new constructs will be useful to better understand SRP function in bacteria.

File Format

application/pdf

Included in

Microbiology Commons

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Apr 1st, 12:00 AM

Determining the cellular localization of the bacterial signal recognition particle by the use of fluorescent protein gene fusions

The signal recognition particle (SRP) is a highly conserved molecular machine that targets hydrophobic polypeptides to cellular membranes of both eukaryotes and prokaryotes. Escherichia coli is an attractive system to study the system since the SRP is composed of a single protein (Ffh) and a small structural RNA (4.5S RNA). While much is known about the biochemical activity of the SRP in E. coli, how the cellular localization of Ffh and 4.5S RNA influences their activity is not as well understood. To approach this question, we have used green fluorescent protein (GFP) and the flavin-based fluorescent protein iLOV to tag the Ffh protein at both the amino- and carboxy-termini. In addition, we have also used the Broccoli fluorescent RNA to localize 4.5S RNA within living cells. Upon completion of tests to observe each construct’s ability to complement an ffh deletion mutation, we discovered that fluorescent tagging altered the function of the Ffh protein. In characterizing the gene fusions it was observed that GFP fusions to either the amino- or carboxy terminus resulted in a dominant negative phenotype. While the fluorescent tags did interfere with protein function, the new constructs will be useful to better understand SRP function in bacteria.