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.

Johansson HE, Dertinger D, LeCuyer KA, Behlen LS, Greef CH, Uhlenbeck OC. (1998) A thermodynamic analysis of the sequence specific binding of RNA by MS2 coat protein. PNAS95, 9244-9249.

Dertinger D, Behlen LS, Uhlenbeck OC. (2000) Using phosphorothioate substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex. Biochemistry 39, in press.

Hertel KJ, Stage-Zimmermann TK, Ammons G, Uhlenbeck OC. (1998) Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme. Biochemistry 37, 16983-16988.

Stage-Zimmermann TK, Uhlenbeck OC. (1998) Hammerhead ribozyme kinetics. RNA 4, 875-889.

Feig AL, Panek M, Horrocks WD Jr, Uhlenbeck OC. (1999) Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme by luminescence spectroscopy. Chemistry & Biology 6, 801-810.

Derrick WB, Greef CH, Caruthers, MH, Uhlenbeck OC. (2000) Hammerhead cleavage of the phosphorodithioate linkage. Biochemistry 39, in press.

Pleiss JA, Derrick ML, Uhlenbeck OC. (1998) T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates. RNA 4, 1313-1317.

Wolfson AD, Pleiss, JA, Uhlenbeck OC. (1998) A new assay for tRNA aminoacylation kinetics. RNA 4, 1019-1023.

Dreher TW, Uhlenbeck OC, Browning KS. (1999) Quantitative assessment of EF-1a GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNAs and tRNA shows close correspondence to the RNA binding properties of EF-Tu. J. Biol. Chem. 274, 666-672.

Tsu CA, Uhlenbeck OC. (1998) Kinetic analysis of the RNA-dependent ATPase activity of DbpA, an E. coli DEAD protein specific for 23S rRNA. Biochemistry 37, 16989-16996.

Kossen K, Uhlenbeck OC. (1999) Cloning and biochemical characterization of B. subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: Delineation of a novel sub-family of bacterial DEAD proteins. Nucleic Acids Res. 27, 3811-3820.

View all publications by publications by Olke C. Uhlenbeck listed in the National Library of Medicine (PubMed).