Abstract

The overall goals of this project are to study the dynamics of ribosome movement along mRNA, the penultimate step in protein assembly, and to progress towards a clearer understanding of the processes that control this movement. Knowledge of the details of protein assembly will further the development of genetically based pharmaceuticals and thereby improve human health. Towards this end we will use total internal reflection fluorescence (TIRF) video microscopy to study folding dynamics of mRNA in real time using nanometer-resolved localization of quantum dot fluorescent DMA probes placed strategically along the mRNA. To vary the dynamics microfluidic systems will be used for precise delivery of reagents and for accurate control of temperature. A novel trip-wire probe methodology will be used to study ribosome/mRNA interactions. Quantum dot probes conjugated to short mRNA sequences will be dislodged as the ribosome passes over them. The resulting loss of fluorescence establishes position and allows precise time measurement with 0.1 sec resolution. Multiple probes placed strategically along the mRNA will allow detailed examination of ribosome/RNA interactions. With the multiple probe technology we will study the effect of mRNA secondary and tertiary structure on ribosome processivity and translation kinetics. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037006-18
Application #
7162179
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Lewis, Catherine D
Project Start
1986-07-01
Project End
2009-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
18
Fiscal Year
2007
Total Cost
$248,327
Indirect Cost

  Institution

Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109

  Publications

Rhee, Minsoung; Burns, Mark A (2009) Microfluidic pneumatic logic circuits and digital pneumatic microprocessors for integrated microfluidic systems. Lab Chip 9:3131-43
Al-Hashimi, Hashim M; Walter, Nils G (2008) RNA dynamics: it is about time. Curr Opin Struct Biol 18:321-9
Walter, Nils G; Huang, Cheng-Yen; Manzo, Anthony J et al. (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475-89
Rhee, Minsoung; Burns, Mark A (2008) Microfluidic assembly blocks. Lab Chip 8:1365-73
Rhee, Minsoung; Burns, Mark A (2008) Drop mixing in a microchannel for lab-on-a-chip platforms. Langmuir 24:590-601
Walter, Nils G (2007) Ribozyme catalysis revisited: is water involved? Mol Cell 28:923-9
Yamaguchi, Yoshinori; Todorov, Todor I; Morris, Michael D et al. (2004) Distribution of single DNA molecule electrophoretic mobilities in semidilute and dilute hydroxyethylcellulose solutions. Electrophoresis 25:999-1006
Todorov, Todor I; Yamaguchi, Yoshinori; Morris, Michael D (2003) Effect of urea on the polymer buffer solutions used for the electrophoretic separations of nucleic acids. Anal Chem 75:1837-43
Todorov, Todor I; Morris, Michael D (2002) Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions. Electrophoresis 23:1033-44
de Carmejane, O; Yamaguchi, Y; Todorov, T I et al. (2001) Three-dimensional observation of electrophoretic migration of dsDNA in semidilute hydroxyethylcellulose solution. Electrophoresis 22:2433-41

Showing the most recent 10 out of 43 publications