BBMB Seminar, May 19th

DEPARTMENT OF BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY

SEMINAR SERIES SPRING 2011

 

Ilchung Shin

Department of Biochemistry, Biophysics and Molecular Biology

Iowa State University

“Imaging gene expression in real-time using aptamers”

Reporter proteins such as fluorescent proteins and luciferase allow for non-invasive detection of the products of gene expression in living cells. However, current reporter systems do not provide for real-time imaging of promoter activity in living cells. This is because of the long time after transcription required for RNA maturation, protein synthesis and maturation. We have developed an RNA reporter system for imaging in real-time to immediately detect changes in promoter activity. The RNA reporter uses strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer Genetic tags), which can be expressed from a promoter of choice. The tobramycin, neomycin and PDC RNA aptamers have been utilized for this system and expressed in yeast from the GAL1 promoter. For imaging, the yeast cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal to noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after activation of promoter by galactose. For the application of IMAGEtags, transcriptional initiation and elongation rate of RNA polymerase II (RNAPII) have been measured because transcriptional initiation and elongation are two regulatory steps in transcription. To identify transcriptional initiation, the FRET signal was measured from ligands bound to IMAGEtags synthesized in response to activation ofthe GAL1 promoter by galactose. IMAGEtags were also utilized as markers to measure the elongation rate of RNAPII in vivo and in real-time. For this measurement, IMAGEtags were inserted in the initial and/or terminal regions of YLR454 gene after the GAL1 promoter. The timing of the appearance of the FRET signals from the ligand interactions with the IMAGEtags were used to monitor RNAPII passage through defined positions in the gene as it was transcribed in living yeast cells.

Thursday, April 28, 2011 4:10 p.m. 1414 Molecular Biology Building

HOST: Marit Nilsen-Hamilton

PLEASE JOIN US AT 3:45 IN THE MBB ATRIUM FOR REFRESHMENTS