Paul Wollenzien

Professor

University of California, Berkeley, CA Ph.D, 1978 Physical Chemistry
University of Wisconsin, Madison, WI B.S., 1970 Chemistry

E-mail: paul_wollenzien@ncsu.edu
Office: 340 Polk Hall
Phone: (919) 515-5703

Research: Ribosome structure and function

Ribosomes perform the fundamental biochemical process of protein synthesis in a highly conserved way in all organisms. The structure of both ribosomal subunits determined by x-ray crystallography structures indicate that the important active sites are composed of rRNA elements. It has been proposed for some time that conformational changes in the ribosome accompany many important steps in protein synthesis. However, this has been difficult to investigate because of the absence of techniques that can determine and monitor higher order structure in large RNAs. We have developed techniques to evaluate UV irradiation-induced RNA-RNA crosslinking that allow us to determine intramolecular distances and distance changes in the ribosome. As a complementary approach, photochemical reagents (psoralens, psoralen derivatives and 4-thiouridine) have been targeted to specific sites in the rRNA prior to reconstitution into 30S ribosomal subunits. These have been characterized for their functional activity and structure and they can be used in the same experiments as native subunits.

We have determined conformational changes in the 16S rRNA in, and around, the decoding region during ribosome subunit association, titration with Mg+2, during association with initiation factor IF3, and in response to tRNA binding to the P-site. We speculate that these structure changes are related to changes in the ribosome that occur during the movement of the tRNA during the elongation cycle. Experiments with initiator tRNA and IF3 are also consistent with information that the 30S pre-initiation complex contains initiator tRNA bound in a stand-by site different than the P site. In another experiment, we have detected specific tertiary structure changes upon the binding of some antibiotics known to interact with the 30S subunit. These experiments provided new distance constraints that are useful in constructing and testing three dimensional models and they also demonstrate that the approach we are taking has the sensitivity to reveal higher order changes in the rRNA during translation.

Selected Publications:

Huggins, W., Shapkina, T. and Wollenzien, P. (2007) Conformational energy and structure in canonical and non-canonical forms of tRNA determined by temperature analysis of the rate of s4U8-C13 photocrosslinking, RNA, in press.

Huggins, W., Ghosh, S., Shapkina, Nanda, K. and Wollenzien, P. (2007) RNA-RNA photocrosslinking is restricted by hydrogen bonding and molecular packing density in the 16S ribosomal RNA in the 30S subunit. Biophysical Journal, in review.

Huggins, W., Ghosh, S., Nanda, K. and Wollenzien, P. (2005) Internucleotide movements during formation of 16S rRNA-rRNA photocrossslinks and their connection to the 30S subunit conformational dynamics, J. Mol. Biol., 354, 358-74.

Huggins, W. and Wollenzien, P. (2004) A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the ribosome, Nucl. Acids Res. 32, 6548-6556.

Nanda, K., and Wollenzien, P. (2004) Pattern of 4-thiouridine induced crosslinking in 16S ribosomal RNA in the Escherichia coli 30S subunit. Biochemistry, 43, 8923-8934.

Shapkina, T., Lappi, S., Franzen, S., and Wollenzien, P. (2004) Efficiency and pattern of UV pulse laser induced RNA-RNA crosslinking in the ribosome. Nucl. Acids Res. 32, 1518-1526.

Noah, J.W. Shapkina, T., Nanda, K., Huggins, W. and Wollenzien, P. (2003) Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P and P/E-site tRNAPhe binding. Biochemistry, 42, 13386-14396.

Zhirnov, O. V. and Wollenzien, P. (2003) Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome. Photochemistry and Photobiological Sciences 2, 688-693.

Dolan, M.A., Babin, P. and Wollenzien, P. (2001) Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modelling. Jour. Mol. Graphics Modeling. 19: 495-513.

Juzumiene, D.I., and Wollenzien, P. (2001) Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking. RNA. 1: 71-84.

Noah, J.W., Shapkina, T.G. and Wollenzien, P. (2000). UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry.Nucl. Acids Res.28, 3785-3792.

Mundus, D. and Wollenzien, P. (2000). Addition of site specific psoralen to large RNAs and their use in directed crosslinking. Methods Enzymol. 318, 104-118.

Shapkina, T., Dolan, M.A., Babin, P. and Wollenzien, P. (2000). Structural effects initiation factor IF3 on the 16S rRNA structure in complexes with mRNA and tRNAfMet. J. Mol. Biol.299, 615-628.

Juzumiene, D., and Wollenzien, P. (2000). Three dimensional organization of the 16S rRNA around its 5' terminus region determined by site directed crosslinking. RNA,6, 26-40.

Babin, P., Dolan, M., Wollenzien, P. and Gutell, R.R. (1999). Identity and geometry of a base triple in 16S rRNA determined by sequence comparison and molecular modeling. RNA, 5, 1430-1439.

Noah, J.W., Dolan, M., Babin, P., and Wollenzien, P. (1999). Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30S ribosomal subunit. J. Biol. Chem. 274, 16577-16581.

 

 

 Supplemental Material:

Supplemental material for Huggins, W., Shapkina, T. and Wollenzien, P. 2007. Conformational energy and structure in canonical and non-canonical forms of tRNA determined by temperature analysis of the rate of s4U8-C13 photocrosslinking, RNA, in press.  Link


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