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The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions1

https://doi.org/10.1006/jmbi.1997.1144Get rights and content

Abstract

Crystal structures of two complexes between recombinant MS2 capsids and RNA operator fragments have been determined at 2.7 Å resolution. The coat protein of the RNA bacteriophage MS2 is bifunctional; it forms the icosahedral virus shell to protect the viral nucleic acid and it acts as a translational repressor by binding with high specificity to a unique site on the RNA, a single stem-loop structure, containing the initiation codon of the gene for the viral replicase. In order to determine the structure of these protein-RNA complexes, we have used chemically synthesized variants of the stem-loop fragment and soaked them into crystals of recombinant capsids. The RNA stem-loop, as bound to the protein, forms a crescent-like structure and interacts with the surface of the β-sheet of a coat protein dimer. It makes protein contacts with seven phosphate groups on the 5′ side of the stem-loop, with a pyrimidine base at position −5, which stacks onto a tyrosine, and with two exposed adenine bases, one in the loop and one at a bulge in the stem. Replacement of the wild-type uridine with a cytosine at position −5 increases the affinity of the RNA to the dimer significantly. The complex with RNA stem-loop having cytosine at this position differs from that of the wild-type complex mainly by having one extra intramolecular RNA interaction and one extra water-mediated hydrogen bond.

Introduction

In contrast to DNA binding proteins, very little is known about sequence-specific protein-RNA interactions. To date, only five structures of protein-RNA complexes are known; three amino-acyl-tRNA synthetases complexed with their cognate tRNAs Rould et al 1991, Cavarelli et al 1993, Biou et al 1994, the RNA-binding domain of the U1A spliceosomal protein bound to hairpin II of U1 small nuclear RNA (Oubridge et al., 1994) and the bacteriophage MS2 coat protein complexed with an RNA stem-loop (Valegård et al., 1994).

The simple RNA bacteriophages of Escherichia coli have long been used as a model system for the study of genetic control at a molecular level. The bacteriophages are divided into four different groups based on serological cross-reactivity, replicase specificity and physical properties (Fiers, 1979). The most commonly studied are MS2 and R17 (group I), GA (group II), Qβ (group III) and SP (group IV). In the case of MS2, the particles contain a single-stranded mRNA molecule of 3569 nucleotides (Fiers et al., 1976), a maturation protein of 393 amino acids, and 180 identical coat protein molecules of 129 amino acids arranged in icosahedral T = 3 symmetry (Fiers, 1979).

The three-dimensional structure of the intact MS2 virus has been determined and refined at 2.8 Å resolution Valegard et al 1990, Golmohammadi et al 1993. The monomer of the coat protein is built up of seven β-strands and two α-helices, where five of these strands form a sheet with a β-meander topology. The icosahedral asymmetric unit contains three subunits; A, B and C (Figure 1). The main conformational difference between the three chemically identical monomers is in the 15 residue loop between the F and G strands, the FG loop, which makes either a fivefold or a quasi-sixfold contact. The FG loops of the A and C subunits are very similar and have an extended conformation while in the B subunit the FG loop is folded back and has a closer contact with the rest of the protein. The basic building block of the virus shell is the compact coat protein dimer. The 90 dimers have two distinct but similar conformations: the C/C conformation at the 2-fold axis and the A/B conformation at the quasi-2-fold axis. In the dimer, ten β-strands form a twisted β-sheet that faces the interior of the virus and the RNA.

The coat proteins of the MS2-related phages work as translational repressors by binding to a site on the viral RNA containing the ribosome binding site for the replicase and its initiation codon. The operator sequence has been studied extensively as a model of sequence-specific RNA-protein interactions (Witherell et al., 1991). It has been shown that the coat protein, as a dimer, binds specifically to a small stem-loop structure containing the initiation codon Gott et al 1993, Stockley et al 1995. The proposed secondary structure of the stem-loop in the viral RNA consists of a lower stem of five base-pairs, a bulged purine, an upper stem of two base-pairs, and a four-nucleotide loop (Figure 2(a)). Only one of the 19 nucleotides requires exact identity for tight binding. The conserved nucleotide is an adenine at position −4. Furthermore for tight binding, the nucleotide at position −5 must be a pyrimidine and the bulged base in the stem and the nucleotide at position −7 in the loop a purine (Witherell et al., 1991). Deletion of the purine at position −10 eliminates protein binding Romaniuk et al 1987, Wu and Uhlenbeck 1987, Talbot et al 1990.

We have used chemically synthesized variants of the stem-loop fragment and soaked these into crystals of recombinant MS2 capsids (Figure 2(a)). The virus capsid contains large holes at both the 5-fold and the 3-fold axes which probably enable entry of these RNA fragments. The crystal structure at 3.0 Å resolution of a complex between MS2 capsids and a 19 nucleotide long RNA fragment with a substitution of the wild-type uridine at position −5 by cytidine, has been published (Valegård et al., 1994). This variant operator binds more tightly to the coat protein than the wild-type sequence. In order to identify the structural differences responsible for this difference in affinity, we have now determined and refined the structures of this complex and the wild-type complex at 2.7 Å resolution.

Section snippets

Quality of the models

Crystallographic details and statistical information on the structure determinations and refinements of the MS2/wild-type RNA complex (wild-type complex) and MS2/C-5 variant complex (C-5 complex) are given in Materials and Methods.

The quality of the electron density map for the RNA is shown in Figure 2, Figure 3. The level of the electron density for the RNA fragments is comparable to that of the protein subunits. The electron density map was clear for all residues and nucleotides except for

Synthesis, crystallization and data collection

The MS2 coat protein gene has been cloned and overexpressed in E. coli (Mastico et al., 1993). The coat protein assembles spontaneously into phage-like particles which can be isolated and purified by size exclusion chromatography. Recombinant capsids of the bacteriophage MS2 is composed of 180 coat protein molecules arranged in icosahedral T = 3 symmetry. Crystals of the recombinant capsids were grown by vapour diffusion from hanging drops containing 0.6 mg ml−1 capsid, 1.25% (w/v)

Acknowledgements

We thank Kerstin Fridborg and Roshan Golmohammadi for help with data collection and the staff at the synchrotrons in Daresbury, Hamburg and Grenoble. This work was supported by grants from the Swedish Natural Science Research Council, the UK SERC and The Wellcome Trust. The atomic coordinates have been deposited in the Brookhaven Protein Data Bank (entry number 1zdi and 1zdh).

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    Present addresses: K. Valegård, Department of Biochemistry, Uppsala University, Box 576, S-751 23 Uppsala, Sweden; S. van den Worm, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, NL-2300 RA Leiden, Netherlands.

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