Wali Karzai, Ph. D. Associate Professor of Biochemistry Department of Biochemistry and Cell Biology And Center for Infectious Diseases Stony Brook University Phone: (631) 632-1688 Fax: (631) 632-8575 E-mail: akarzai@ms.cc.sunysb.edu
	Research Description

The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and an mRNA. The tmRNA, along with its protein cofactors, orchestrates three key biological functions: 1) recognition and rescue of ribosomes stalled on aberrant mRNAs, 2) disposal of the causative defective mRNAs, and 3) addition of a degradation tag to ribosome-associated protein fragments for directed proteolysis. Although not essential in E. coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence suggests that in addition to its quality control function the tmRNA system might also play a key regulatory role in certain physiological pathways. A major research focus in my lab concerns the SmpB•tmRNA quality control system (see Figure below).

CURRENT MODEL FOR tmRNA FUNCTION:
In this model, SmpB protein binds tmRNA and the complex is aminoacylated by Ala-RS (a). The aminoacylated-tmRNA-SmpB complex is recognized by EF-Tu-GTP (b) to form a stalled ribosome recognition complex. Ribosomes stalled at the 3’-end of nonstop mRNAs are initially recognized by this quaternary-complex in a pre-accommodated state (c). Proper accommodation of the tRNA-like domain of tmRNA into the ribosomal A-site is then triggered by contacts, perhaps elicited by the C-terminal tail of SmpB, that promote GTP hydrolysis on EF-Tu. Accommodation is followed by the first transpeptidation reaction that links the alanine charge of tmRNA to the incomplete polypeptide (d). tmRNA then switches from a tRNA-like mode to an mRNA-like mode, engaging the ribosome on its own peptide reading frame (e), concomitant with the displacement of the defective mRNA (f). The damaged mRNA is selectively recognized and degraded by RNase R, in an SmpB and tmRNA dependent manner. The rescued ribosome resumes translation with the tmRNA ORF as its surrogate template (g). Translation terminates on the tmRNA-encoded stop codon, permitting recycling of stalled ribosomes into the cellular pool (h). The nascent polypeptide, now marked with an 11-amino acid degradation tag, is released from the translation machinery (i), and specifically recognized and degraded by C-terminal specific cellular proteases (j).

We are also interested in understanding how sequence and structure in RNA-binding proteins contribute to the formation of specific RNA-protein complexes and how these complexes promote specific biological functions. We use a combination of protein biochemistry, functional genomics, bioinformatics, and X-ray crystallography to determine the biological function and mechanism of action of specific RNA-protein complexes.

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