Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding.
Transfer RNA structure involves complex folding interactions of the TPsiC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TPsiC domain, rT54, Psi55 and m5C49, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA(Phe) T-half molecules (nucleosides 40-72) with the corresponding unmodified D-half molecule (nucleosides 1-30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg2+ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K(d)s (rT54 = 6 +/- 2, m5C49 = 11 +/- 2, Psi55 = 14 +/- 5, and rT54,Psi55 = 11 +/- 3 microM) significantly lower than that of the unmodified T-half (40 +/- 10 microM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA(Phe) and native yeast tRNA(Phe), as determined by lead cleavage patterns at U17 and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA's TPsiC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.
Nobles
Kelly N
KN
Department of Molecular and Structural Biochemistry, North Carolina State University, 128 Polk Hall, PO Box 7622, Raleigh, NC 27695-7622, USA.
Yarian
Connie S
CS
Liu
Guihua
G
Guenther
Richard H
RH
Agris
Paul F
PF
D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D006860:Hydrogen Bonding; D007854:Lead / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism*; D020871:RNA Stability / Q000187:drug effects; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D013816:Thermodynamics
D009705:Nucleosides; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D007854:Lead; D006163:Ribonuclease T1; D008274:Magnesium