Local folding coupled to RNA binding in the yeast ribosomal protein L30

doi:10.1006/jmbi.1999.3044

Journal of Molecular Biology

Volume 292, Issue 2, 17 September 1999, Pages 345-359

https://doi.org/10.1006/jmbi.1999.3044 Get rights and content

Abstract

The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regulates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in the free (f L30) and the pre-mRNA bound (b L30) forms have been solved by nuclear magnetic resonance spectroscopy. Both protein structures contain four alternating α-helices and four β-strands segments and adopt an overall topology that is an αβα three-layer sandwich, representing a unique fold. Three loops on one end of the αβα sandwich have been mapped as the RNA binding site on the basis of structural comparison, chemical shift perturbation and the inter-molecular nuclear Overhauser effects to the RNA. The structural and dynamic comparison of f L30 and b L30 reveals that local dynamics may play an important role in the RNA binding. The fourth helix in b L30 is longer than in f L30, and is stabilized by RNA binding. The exposed hydrophobic surface that is buried upon RNA binding may provide the energy necessary to drive secondary structure formation, and may account for the increased stability of b L30.

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

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Journal of Molecular Biology

Regular articleLocal folding coupled to RNA binding in the yeast ribosomal protein L301

Abstract

Introduction

Section snippets

Protein binding and CD analysis

Structural comparison between the free and RNA-bound protein

Conclusion

Protein expression and purification

Acknowledgements

J. Mol. Graph

J. Biol. Chem

Structure

J. Magn. Reson. ser. A

Cell

J. Magn. Reson

J. Mol. Biol

J. Mol. Biol

J. Magn. Reson. ser. B

J. Mol. Biol

Structure

J. Magn. Reson. ser. B

J. Mol. Biol

J. Magn. Reson. ser. B

J. Mol. Biol

Cell

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

Regular article
Local folding coupled to RNA binding in the yeast ribosomal protein L30¹

Assignment of the side-chain¹H and¹³C resonances of interleukin-1β using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy