RNA

D. Söll, S. Nishimura, P. Moore

• Verlag: Pergamon
• Jahr: 2001
• ISBN:
  9780080540627
• Thema: Biowissenschaften
• Thema: Biochemie

This publication summarizes the current status of our understanding of RNA, with particular emphasis on the chemistry of this key biological molecule. The various RNAs covered are messenger RNA, ribosomal RNA, transfer RNA and RNA enzymes (ribozymes). The different chapters detail biophysical and chemical methods to investigate RNA structure and function, the synthesis of native and modified RNAs and the latest advances in our understanding of the vast array of biological processes in which RNA is involved.

1

A Spectroscopist’s View of RNA Conformation: RNA Structural Motifs

Peter B. Moore Yale University, New Haven, CT, USA

1.1 INTRODUCTION 2

1.2 THE DETERMINATION OF RNA STRUCTURES BY NMR 2

1.2.1 NMR Fundamentals 2

1.2.2 Chemical Shift 3

1.2.3 Couplings and Torsion Angles 3

1.2.4 Spin–Lattice Relaxation: Nuclear Overhauser Effects and Distances 4

1.2.5 Spin–Spin Relaxation: Molecular Weight Limitations 5

1.2.6 Samples 6

1.2.7 Multidimensional NMR 6

1.2.8 Assignments 6

1.2.9 Helices and Torsion Angles 7

1.2.10 Distance Estimation 8

1.2.11 Structure Calculations 9

1.3 SOLUTION STRUCTURES AND CRYSTAL STRUCTURES COMPARED 9

1.3.1 On the Properties of Crystallographic Structures 9

1.3.2 Solution Structures 10

1.3.3 Constraints and Computations 10

1.3.4 Experimental Comparisons of Solution and Crystal Structures 11

1.3.5 New Approaches 12

1.4 LESSONS LEARNED ABOUT MOTIFS BY NMR 12

1.4.1 RNA Organization in General 13

1.4.2 Terminal Loops 13

1.4.2.1 U-turns 13

1.4.2.2 Tetraloops 14

1.4.2.3 Other terminal loops 15

1.4.3 Internal Loops 15

1.4.3.1 Symmetric internal loop motifs 15

1.4.3.2 Asymmetric internal loop motifs 16

1.4.4 Pseudoknots 17

1.5 REFERENCES 17

1.1 INTRODUCTION

Biologically, RNA mediates between DNA and protein — DNA makes RNA makes protein — and RNA is also intermediate between DNA and protein chemically. Some RNAs are carriers of genetic information, like DNA, and others, e.g. transfer RNAs and ribosomal RNAs, are protein-like. Their functions depend on their conformations as much as their sequences, and some even have enzymatic activity.

Even though RNA biochemists have recognized their need for structures almost as long as protein biochemists, far more is known about proteins than RNAs. Coordinates for over 8000 proteins have been deposited in the Protein Data Bank, but the number of RNA entries is of the order of 100, and many of them describe RNA fragments, not whole molecules.

All the RNA structures available before 1985 were crystal structures, and X-ray crystallography remains the dominant method for determining RNA conformation. By the late 1980s, nuclear magnetic resonance (NMR) had emerged as a viable alternative, but for many years, only a few structures a year were being solved spectroscopically. In the last two years, the production rate has risen to roughly a structure a month, and because the field is taking off, it is time RNA biochemists understand what the structures spectroscopists provide are all about.

This chapter describes how RNA conformations are determined by NMR. The description provided is intended to help biochemists understand what NMR structures are, not to teach them how to do it. The chapter also summarizes what NMR has taught us about RNA motifs. For these purposes, a motif is any assembly of nucleotides bigger than a base triple that has a distinctive conformation and is common in RNAs.

1.2 THE DETERMINATION OF RNA STRUCTURES BY NMR

The behavior of all atoms that have non-zero nuclear spins can be studied by NMR, and the predominant isotopes of two of the five elements abundant in RNA qualify in this regard: ¹H and ³¹ P. Both have spins of ½. Those not content with the information ¹H and ³¹P spectra provide, can easily prepare RNAs labeled with ¹³C and/or ¹⁵N, which are also spin-½ nuclei (see below). Thus NMR spectra can be obtained from all the atoms in a nucleic acid except its oxygens, for which no suitable isotope exists. What can be learned from them?

The answer to this question, of course, can be mined out of the primary NMR literature, but it is vast and much of it too technical for non-specialists. For that reason, rather than fill this chapter with references its intended readers will find useless, I direct them here to a few secondary sources. For NMR fundamentals, Slichter’s book is excellent.¹ It is complete, and its verbal descriptions are good enough so that readers need not wade through its (many) derivations. Those interested in multidimensional NMR, about which almost nothing is said below, can consult Goldman’s short monograph,² or the treatise of Ernst and coworkers.³ Although a bit dated at this point, Wüthrich’s book on the NMR of proteins and nucleic acids is so useful the cover has fallen off the local copy.⁴ A more technically oriented text on protein NMR appeared recently, which is also useful.⁵

1.2.1 NMR Fundamentals

Nuclei that have spin (and not all do) have intrinsic magnetic moments, and thus orient like compass needles when placed in magnetic fields. Because nuclei are very small, their response is quantized. Spin-½ nuclei orient themselves in magnetic fields in only two ways: parallel to it or antiparallel to it. Because the energy associated with the parallel orientation is only slightly lower than that of the antiparallel orientation, the number of nuclei in the parallel orientation is only slightly larger than the number in the antiparallel orientation in any population of magnetically active atoms that has come to equilibrium in a magnetic field. In the strongest available magnets, the excess is only a few per million. The tiny bulk magnetization their collective alignment produces is what NMR spectroscopists study. Sensitivity is not one of NMR’s selling points!

An NMR spectrometer consists of a magnet to orient the nuclei in samples, a radio frequency transmitter to perturb nuclear orientations in controlled ways, and a receiver to detect the electromagnetic signals generated when the orientations of the magnetic moments of aligned populations of nuclei are perturbed. NMR spectrometers produce spectra, which are displays of the magnitude of these electromagnetic signals as a function of perturbing frequency. A peak in such a display is a resonance.

1.2.2 Chemical Shift

Electromagnetic radiation causes the reorientation of spin-½ nuclei that have become aligned in external magnetic fields most efficiently when the product of Planck’s constant and the frequency of the reorienting radiation equals the difference in energy between their two possible orientations, i.e. hv = ∆E. That frequency, the resonant frequency, is the one at which the intensity of a resonance in a spectrum is maximum. The energy difference that determines a resonant frequency is the product of the strength of the orienting magnetic field a nucleus experiences and its intrinsic magnetic moment.

The magnetic moments of all the nuclides relevant to biochemists were measured long ago, and they differ so much that there is no possibility of confusing the resonances of one species with those of another, a hydrogen resonance with a phosphorus resonance, for example. (N.B.: The resonant frequency of protons in a 500 MHz NMR spectrometer is 500 MHz.)

The reason NMR interests chemists is that the magnetic field a nucleus experiences, and hence the frequency at which it resonates, depends on its chemical context. Nuclei in molecules are surrounded by electrons, which for these purposes are best thought of as particles in continual motion. When a charged particle moves through a magnetic field, a circular component is added to its trajectory, and charged particles moving in circles generate magnetic fields. Thus when a molecule is placed in a magnetic field, it becomes a tiny solenoidal magnet the field of which (usually) opposes the external field. As you would expect, both the direction and the strength of the field induced in a molecule this way depend on its structure and on its orientation with respect to the inducing field. In solution, where rapid molecular tumbling leads to averaging, orientation effects disappear, and the atom-to-atom variations in the strength of the induced magnetic field within a molecule are reduced to a few millionths the magnitude of the inducing field. Small though these induced field differences are, the contribution they make to the total field experienced by each nucleus is easily detected because the receivers in modern NMR spectrometers have frequency resolutions of about 1 part in 10⁸. Thus the proton spectrum of a biological macromolecule is a set of resonances differing modestly in frequency, not a single, massive resonance. Incidentally, all else being equal, the magnitude of each resonance produced by a sample is proportional to the number of nuclei contributing to it.

The frequency differences that distinguish resonances...