Faculty
Olke C. Uhlenbeck, PhD
Professor
Molecular Biosciences
PhD, Harvard
Email: o-uhlenbeck@northwestern.edu
Phone: (847) 491-5139
Fax: (847) 491-5444
Room: Cook Rm 4143
Research Interests
RNA Biochemistry
MS2 Phage Coat Protein - RNA Interaction
This system is being studied for several reasons: (1) it is an example of a sequence-specific RNA-protein interaction, (2) it participates in a well-behaved in vitro capsid assembly reaction, and (3) it is a good model system to study how protein finds a target on a large RNA molecule. Available are an X-ray crystal structure of the RNA-protein complex and an NMR structure of the free RNA hairpin target. Current efforts focus on understanding how the thermodynamic details of sequence-specific "recognition" is achieved. We have made mutations in all the amino acids believed to make contact with the RNA and are evaluating the affinity of the mutant proteins to the normal RNA target as well as to targets that have single atom changes in either the bases or the phosphodiester backbone. It is already clear that nearly all the contacts predicted by the co-crystal structure contribute to the total free energy of binding. Thus, unlike several protein-protein interfaces that have been analyzed in a similar way, there are no "hot spots" that dominate the affinity. However, we have several examples where affinity and specificity are defined by structural elements of the RNA in its free form.
The Hammerhead Ribozyme
This 35 to 45 nucleotide RNA motif derived from a viroid undergoes autocatalytic cleavage at a unique site to give 2' 3' cyclic phosphate and a 5' hydroxyl termini. It is considerably smaller than any of the other known catalytic RNAs and is, therefore, particularly amenable structure-function and mechanistic studies. It has also been proposed to be a useful gene therapy reagent. Considerable effort has been spent developing a kinetic and thermodynamic framework for the cleavage reaction and studying the cleavage properties of RNAs containing single atom changes. In the past several years, efforts has focused on finding the sites of catalytically important divalent metal ions and understanding their role in catalysis. We are currently examining the consequences of making site specific crosslinks within the hammerhead. One crosslink has been shown to greatly increase the rate of the reverse, ligation rate without changing the cleavage rate. A recent proposal that the hammerhead undergoes a large conformational change prior to catalysis is being tested by searching for crosslinks that increase the forward reaction rate.
The Recognition of tRNA
Our long standing interest in the interaction of tRNA with various components of the translation apparatus is currently focused in two areas. (1) We have developed a new assay for aminoacyl tRNA synthetases that permits us to follow the attachment of the amino acid onto tRNA under pre-study state (enzyme excess) conditions. This will allow us to examine the reaction in a way that more closely reflects the physiological situation and evaluate the activity of modified tRNAs in a more meaningful fashion. (2) We have prepared a series of derivatives of yeast tRNA Phe which contain single deoxynucleotides and evaluated their ability to bind elongation factor Tu. Of the eight 2' hydroxyl groups predicted by the crystal structure to make contact with the protein, only four have a thermodynamic effect when changed to a deoxynucleotide. We are currently evaluating why the other four do not show an effect. In addition, there is one 2' hydroxyl that affects protein binding by stabilizing the structure of the tRNA in its bound form. This same set of 2' hydroxyl modified tRNAs have revealed that EF-1a, the eukaryotic homologue of EF-Tu, interacts with tRNA in a similar but distinct manner. In the future, we will examine the activity of these modified tRNAs with other enzymes that interact with tRNA, including the ribosome.
E. coli DbpA.
DEAD/H proteins is a large family of proteins that participate in many different pathways of RNA metabolism. Most DEAD/H proteins are RNA-dependent ATPases and a few show RNA helicase activity. While the exact function of DEAD/H proteins remains unclear, a popular model is that they catalyze the disruption of short RNA helices. We have chosen to work on E. coli DbpA (and its B. subtilis homologue YxiN) because it is unique among DEAD/H proteins in that it binds tightly and with high specificity to a defined target site in E. coli 23S rRNA. It is a very active ATPase with an RNA fragment as short as 23 nucleotides, but shows no ATPase activity without RNA. We have recently shown it is also an RNA helicase. These features make DbpA an ideal candidate for studies to understand the mechanism of this interesting class of proteins.
Selected Publications
A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit. Sharpe Elles LM, Sykes MT, Williamson JR, Uhlenbeck OC. Nucleic Acids Res. 2009 Oct;37(19):6503-14. Epub 2009 Sep 4.
RNA biophysics has come of age. Uhlenbeck OC. Biopolymers. 2009 Oct;91(10):811-4.
Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis. Schrader JM, Chapman SJ, Uhlenbeck OC. J Mol Biol. 2009 Mar 13;386(5):1255-64.
A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding. Ledoux S, Olejniczak M, Uhlenbeck OC. Nat Struct Mol Biol. 2009 Apr;16(4):359-64. Epub 2009 Mar 22.
Specificity of the ribosomal A site for aminoacyl-tRNAs. Dale T, Fahlman RP, Olejniczak M, Uhlenbeck OC. Nucleic Acids Res. 2009 Mar;37(4):1202-10. Epub 2009 Jan 7.
Differential RNA-dependent ATPase activities of four rRNA processing yeast DEAD-box proteins. Garcia I, Uhlenbeck OC. Biochemistry. 2008 Nov 25;47(47):12562-73. Erratum in: Biochemistry. 2009 Feb 3;48(4):810.
Different aa-tRNAs are selected uniformly on the ribosome. Ledoux S, Uhlenbeck OC. Mol Cell. 2008 Jul 11;31(1):114-23.
Catalytic diversity of extended hammerhead ribozymes. Shepotinovskaya IV, Uhlenbeck OC. Biochemistry. 2008 Jul 8;47(27):7034-42. Epub 2008 Jun 11.
Hammerhead redux: does the new structure fit the old biochemical data? Nelson JA, Uhlenbeck OC. RNA. 2008 Apr;14(4):605-15. Epub 2008 Feb 20.
[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation. Ledoux S, Uhlenbeck OC. Methods. 2008 Feb;44(2):74-80.
Minimal and extended hammerheads utilize a similar dynamic reaction mechanism for catalysis. Nelson JA, Uhlenbeck OC. RNA. 2008 Jan;14(1):43-54. Epub 2007 Nov 12.
Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype. Elles LM, Uhlenbeck OC. Nucleic Acids Res. 2008 Jan;36(1):41-50. Epub 2007 Nov 5.
Exploring the specificity of bacterial elongation factor Tu for different tRNAs. Sanderson LE, Uhlenbeck OC. Biochemistry. 2007 May 29;46(21):6194-200. Epub 2007 May 10.
View all publications by Olke C. Uhlenbeck listed in the National Library of Medicine (PubMed). Past and current IBiS students in blue