Journal of Molecular Biology
Regular articleLocal folding coupled to RNA binding in the yeast ribosomal protein L301
Introduction
The yeast Saccharomyces cerevisiae ribosomal protein L30 (MW∼11 kD) (Mager et al., 1997), formerly known as L32 (Dabeva & Warner, 1987), is an essential protein for yeast growth (Dabeva & Warner, 1987). The L30 protein has no homolog in prokarya, but apparently is ubiquitous in eukarya and archaea. The sequence conservation of L30 protein across species in eukarya (Figure 1) implies an important role for the L30 in the ribosome function. However, the rRNA binding site within ribosome is currently not known. The best understood aspect of yeast L30 protein function is the negative feedback regulation of pre-mRNA splicing and mRNA translation upon over-production of L30 Eng and Warner 1991, Li et al 1996. The presence of L30 bound to its pre-mRNA near the 5′ splice site prevents the completion of spliceosome assembly in vitro(Vilardell & Warner, 1994). Binding of L30 close to the translational initiation codon in its mRNA provides another level of auto-regulation (Li et al., 1996). Both splicing and translational regulation involve binding of L30 protein to a consensus sequence in the L30 mRNA that forms an internal loop structure Eng and Warner 1991, Li et al 1996.
Ribosomes are the essential machinery for protein synthesis in all organisms. A typical eukaryotic ribosome contains about 78 different proteins (32 in the 40 S subunit and 46 in the 60 S subunit) (Mager et al., 1997), as compared to some 52 proteins in a prokaryotic ribosome. A surprisingly limited amount of structural information is available for eukaryotic ribosomal proteins, while several structures of prokaryotic ribosomal proteins have been solved recently (Liljas & al-Karadaghi, 1997). As an initial step toward studying the structure and function of the eukaryotic ribosome, it is advantageous to examine individual components. We have successfully prepared pure and active yeast L30 protein by over-expression in Escherichia coli, and solved the high-resolution structure of the protein in free form (f L30) using heteronuclear magnetic resonance spectroscopy. In addition, we have recently completed the structure of the L30-mRNA complex (unpublished results), and for comparison, we present here the bound form of L30 extracted from the complex as b L30. The three-dimensional structures obtained from simulated annealing reveal an αβα three-layer sandwich topology in both f L30 and b L30. Detailed comparisons between the free and bound forms reveal insights to the structure and function of L30 protein. Specifically, side-by side comparisons of chemical shifts, resonance linewidths, and relaxation times for the free and RNA-bound proteins indicate changes in the protein local environment, structure, and dynamics that accompany RNA binding. The L30 structure provides insights into the role of conserved residues in folding of the protein and in RNA binding.
Section snippets
Protein binding and CD analysis
Although yeast L30 is a relatively small protein (MW∼11 kD), previous biochemical studies of the interactions between the L30 and its autoregulatory RNAs were primarily carried out with a 53 kD maltose-binding protein-L30 fusion (MBP-L30) Vilardell and Warner 1994, Li et al 1995. Prior to structural studies using NMR, it is essential to obtain the active L30 protein with a suitable size. Combining the advantages of the MBP-L30 fusion protein for affinity purification and the charge differences
Structural comparison between the free and RNA-bound protein
NMR and CD data both indicate that the f L30 adopts a folded tertiary structure in the absence of its cognate RNA. The NOEs, J-couplings, and secondary chemical shift alignments are similar for most of the free and bound L30. Indeed, the tertiary structures obtained from molecular modeling exhibit a common folding topology, namely, an αβα three-layer sandwich. Based on the overlay of the backbone of f L30 and b L30 average structure (Figure 8(a)), most of the significant structural features are
Conclusion
The yeast L30 is the newest member in the family of eukaryotic ribosomal proteins whose structures have been studied at near-atomic resolution. Significantly, this is the first ribosomal protein whose interaction with its pre-mRNA binding site RNA has been studied using an NMR-based approach. These studies have shown that the L30 protein interacts with its target RNA using three loops at one end of an α/β sandwich. The structure of the free and bound forms of L30 reveal that RNA binding is
Protein expression and purification
The yeast S. cerevisiae ribosomal protein L30 (without the first methionine residue) was over-expressed as a soluble maltose-binding protein fusion (MBP-L30) in E. coli strain JM109, hosting plasmid pMalc-L30 (Vilardell & Warner, 1994). Unlabeled protein was prepared from cells grown in a medium containing 25 g/l of Luria Broth (Gibco) and 100 mg/l ampicillin. Uniformly15N or13C/15N-labeled cells were grown in M9-based minimal medium containing 0.7 g/l (15NH4)2SO4alone or 1.0 g/l (15NH4)2SO4and
Acknowledgements
We thank Dr Susan White at Bryn Mawr College, Dr Jonathan Warner and Dr Joseph Vilardell at Albert Einstein College for providing the pMalc-L30 plasmid. We thank Dr Kwaku Dayie and Dr John Chung at TSRI and Dr Christopher Turner at MIT for their assistance with NMR spectrometers, and Dr Radha Plachikkat for helpful discussions about X-PLOR. We thank Jason Schnell for assistance with X-PLOR to AMBER restraint conversion, and we thank both Jason Schnell and Dr Lena Maler for valuable discussions
References (50)
Ribbon models of macromolecules
J. Mol. Graph
(1987)- et al.
The yeast ribosomal protein L32 and its gene
J. Biol. Chem
(1987) - et al.
Structural evidence for specific S8-RNA and S8-protein interaction within the 30S ribosomal subunitribosomal protein S8 from Bacillus stearothermophilus at 1.9 Å resolution
Structure
(1996) - et al.
Relaxation-rate meausrements for15N-1H groups with pulsed-field gradients and perservation of coherence pathways
J. Magn. Reson. ser. A
(1994) - et al.
Structural basis for the regulation of splicing of a yeast messenger RNA
Cell
(1991) - et al.
A common sense approach to peak picking in two-, three-, and four-dimensional spectra using automatic computer analysis of contour diagrams
J. Magn. Reson
(1991) - et al.
The RNA binding domain of ribosomal protein L11three-dimensional structure of the RNA-bound form of the protein and its interaction with 23S rRNA
J. Mol. Biol
(1997) - et al.
Protein structure comparison by alignment of distance matrices
J. Mol. Biol
(1993) - et al.
A gradient-enhanced HCCH-TOCSY experiment for recording side-chain1H and13C correlations in H2O samples of proteins
J. Magn. Reson. ser. B
(1993) - et al.
Characterization of the pre-mRNA binding site for yeast ribosomal protein L32the importance of a purine-rich internal loop
J. Mol. Biol
(1995)
Thioredoxin-a fold for all reasons
Structure
Gradient-enhanced triple-resonance three-dimensional NMR experiments with improved sensitivity
J. Magn. Reson. ser. B
SCOPa structural classification of proteins database for the investigation of sequences and structures
J. Mol. Biol
Simultaneous acquisition of15N- and 13C-edited NOE spectra of proteins dissolved in H2O
J. Magn. Reson. ser. B
Comparison of super-secondary structures in proteins
J. Mol. Biol
The crystal structure of HaeIII methyltransferase covalently complexed to DNAan extrahelical cytosine and rearranged base pairing
Cell
X-ray structure of the DNase I-d(GGTATACC)2complex at 2.3 Å resolution
J. Mol. Biol
Protein database searches for multiple alignments
Proc. Natl Acad. Sci. USA
Structures of RNA-binding proteins
Quart. Rev. Biophys
X-PLOR (Version 3.1) A System for X-ray Crystallography and NMR
Determination of the helix and beta form of proteins in aqueous solution by circular dichorism
Biochemistry
Crystal structures of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog
Proc. Natl Acad. Sci. USA
Assignment of the side-chain1H and13C resonances of interleukin-1β using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy
Biochemistry
A second generation force field for the simulation of proteins and nucleic acids
J. Am. Chem. Soc
Proteins: Structures and Molecular Principles
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