Contact Information:

c/o HST Division M.I.T.

77 Massachusetts Ave.

Cambridge, MA 02139

phone: 617-253-7608

fax: 617-253-3459

lee_gehrke@hms.harvard.edu

Gehrke Lab Home

Research Summary

The research interests of this laboratory center on understanding the functional significance of RNA structure and RNA-protein interactions in messenger RNA translation, virus replication, and virus assembly. Specific RNA-protein interactions are essential for many biological processes, and disruption of RNA-protein interactions is associated with disease states. However, relatively little is understood about the features of RNAs and proteins that signal the formation of specific ribonucleoprotein complexes.

In this laboratory, the binding of coat protein and peptides to ilarvirus RNAs is used to analyze the molecular details of specific RNA-protein interactions. The ilarviruses are positive-strand RNA viruses that have a number of unusual features that define them as an ideal model for study. The three genomic RNAs plus the subgenomic coat protein RNA are separately encapsidated in bacilliform-shaped particles. Another unusual feature of these viruses is that the viral RNAs alone are not infectious; instead, a few molecules of coat protein or coat protein peptides must be added to activate the early stages of virus replication. Why coat protein is required to initiate replication is not known, but coat protein may regulate plus-strand RNA accumulation and stabilize a 3'-terminal RNA structure that facilitates accurate replication initiation. Another feature of this experimental system is that the coat proteins of the individual ilarviruses are functionally interchangeable for replication, yet unrelated at the primary amino acid sequence level. In other words, even though the coat proteins show little amino acid homology, coat protein from one ilarvirus will specifically bind the RNAs of a different ilarvirus and activate replication. This provides an opportunity to analyze RNA protein interactions in homologous and heterologous combinations of RNAs and coat proteins or peptides with a goal of elucidating a high-resolution structure for an RNA-peptide complex.

A combination of biochemical and biophysical techniques is being used to identify amino acids, nucleotides, and structural elements required for RNA-protein complex formation. An E. coli-based genetic screen permits rapid identification of gain-of-function mutants that suggest nucleotide-amino acid contacts, while electrophoretic mobility shift assays, chemical modification interference studies, hydroxyl radical footprinting, and in vitrogenetic selection are being used for biochemical characterization. To facilitate structural analyses, an ilarvirus coat protein RNA binding domain is being expressed on the surface of cowpea mosaic virus, and the structure of the chimeric virus will be analyzed by X-ray crystallography. NMR analysis of an RNA-peptide complex is also underway.

A second focus of this laboratory is understanding translation-level regulation of gene expression, and the current emphasis is on characterizing the role of 3' untranslated nucleotides of viral and cellular messenger RNAs. Although the 3' untranslated region was previously believed to have little regulatory significance beyond a possible role in RNA stability, recent data suggest roles in RNA localization, regulation of mRNA translational efficiency and stability, and cellular differentiation. The mechanisms, however, are largely unknown. Deletion of the 3' untranslated region of an ilarvirus messenger RNA reduces its translational efficiency both in vitroand in microinjected Xenopus oocytes without affecting messenger RNA stability. This system is being used to probe the cross-talk between 5' and 3' end of messenger RNAs that regulates translational efficiency and competitive activity.

Selected Publications

Ansel-McKinney P, Gehrke L. RNA determinants of a specific RNA-coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs. J Mol Biol. 1998 May 15;278(4):767-85.

Swanson MM, Ansel-McKinney P, Houser-Scott F, Yusibov V, Loesch-Fries LS, Gehrke L. Viral coat protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus RNAs. J Virol. 1998 Apr;72(4):3227-34.

Hann LE, Webb AC, Cai JM, Gehrke L. Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA. Mol Cell Biol. 1997 Apr;17(4):2005-13.

Houser-Scott F, Ansel-McKinney P, Cai JM, Gehrke L. In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3'-terminal AUGC repeats in the viral RNAs. J Virol. 1997 Mar;71(3):2310-9.

Ansel-McKinney P, Scott SW, Swanson M, Ge X, Gehrke L. A plant viral coat protein RNA binding consensus sequence contains a crucial arginine. EMBO J. 1996 Sep 16;15(18):5077-84.