NMR Structure of a Bacteriophage T4 RNA Hairpin Involved in Translational Repression

• Published in: Biochemistry (Easton) 1996-06, Vol.35 (24), p.7664-7674
• Main Authors: Mirmira, Suman R, Tinoco, Ignacio
• Format: Article
• Language: English
• Subjects: Bacteriophage T4 - metabolism; Base Sequence; Consensus Sequence; Hydrogen Bonding; Indicators and Reagents; Magnetic Resonance Spectroscopy - methods; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; Oligoribonucleotides - chemical synthesis; Oligoribonucleotides - chemistry; phage T4; Phosphorus; Protein Biosynthesis; RNA, Viral - chemistry; RNA, Viral - metabolism; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960414y

A high-resolution structure of a 16-nucleotide bacteriophage T4 RNA hairpin, 5‘-GCCU[AAUAACUC]GGGC (loop bases in square brackets), has been determined in solution by proton, phosphorus, and carbon (natural abundance) NMR spectroscopy. This RNA hairpin is known to play a crucial role in the translational repression of bacteriophage T4 DNA polymerase. Ultraviolet absorbance melting curves indicate that the structure formed is unimolecular. The NMR spectra indicate that a single conformation consistent with a hairpin structure is formed. Strong imino−imino NOEs confirm the formation of the G·U base pair at the stem−loop junction. There is no evidence that A5 is protonated (at pH 6.0) and involved in an A+·C pair. However, the NMR data indicate that the stem is extended beyond the G·U pair and that A-form stacking continues for three nucleotides on the 5‘ side and one nucleotide on the 3‘ side. Structure calculations using restraints obtained from NMR data give a precisely defined structure with an average root mean square deviation (RMSD) of approximately 1.2 Å for the entire molecule. The assignment of all the protons and most of the 31P resonances in the loop yielded a large number of distance and torsion angle restraints for these nucleotides. These helped obtain a well-defined loop with an average RMSD of 1.1 Å for the loop nucleotides of 11 converged structures.