Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA.
A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA.
Pleiss
J A
JA
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309, USA.
Uhlenbeck
O C
OC
D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006160:Guanosine Triphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D023282:Nuclease Protection Assays; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012344:RNA, Transfer, Ala / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
D011522:Protons; D012329:RNA, Bacterial; D012344:RNA, Transfer, Ala; D012360:RNA, Transfer, Phe; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D000409:Alanine