Internal dynamics of transfer ribonucleic acid determined by nuclear magnetic resonance of carbon-13-enriched ribose carbon 1.
Carbon-13 enrichment of the C1' position of the ribose moiety in Escherichia coli tRNA has made possible the detailed study of motion in this molecule. Enrichment was accomplished in vivo with a strain, M1R, selected for growth and degree of incorporation of ribose in a stringently defined minimal medium. Purine biosynthesis de novo was blocked with 6-mercaptopurine. Exogenously provided [1-13C]ribose and nucleobases were utilized via the salvage pathway and were required for growth of culture. Carbon-13-enriched transfer RNA in solution at 30 degrees C exhibited a prominent, broad, asymmetric NMR signal at 91.5 ppm for the C1' carbon. Upon heat denaturation of the tRNA, three C1' signals were resolved and could be attributed to the base-specific nucleotides in tRNA: uridine and guanosine at 88.7 ppm; adenosine at 89.5 ppm; and cytidine at 90.6 ppm. Ribose C3' and C5' were partially enriched due to scrambling of ribose carbons in vivo. The minimum net isotopic enrichment of C1' was 33%. Values for the relaxation time T1 and the nuclear Overhauser enhancement (NOE) at 75.5, 67.8, and 25.2 MHz (13C), the NOE at 50.3 MHz, T2 at 75.5 MHz, and line widths over the range of 20-75.5 MHz were analyzed in light of several models for internal motion in macromolecules. The data were inconsistent with physically unreasonable constructs involving free internal diffusion of the C1'-H vector about the glycosidic bond. Internal diffusion (wobble) within a cone or jumps between states were models that did fit the data. For diffusion within a cone, the cone half-angle was 15-20 degrees, with a correlation time of about 2 X 10(-9) s for internal reorientation. With the two-state jump model, the half-angle for jumps about the glycosidic bond was 14 +/- 2 degrees with a lifetime of 2 X 10(-9) s.
D002247:Carbon Isotopes; D055598:Chemical Phenomena; D002621:Chemistry; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial; D012343:RNA, Transfer; D012266:Ribose; D013696:Temperature
D002247:Carbon Isotopes; D012329:RNA, Bacterial; D012266:Ribose; D012343:RNA, Transfer