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Studies of Proteins and RNA-Protein Complexes Involved in Translation and Gene-Expression Regulation
Summary: Maria Garber and her team use the methods of x-ray crystallography and molecular biology to determine the structures of RNA-protein complexes involved in translation and regulation of gene expression. Using structural data, they try to understand the principles underlying macromolecular recognition, with reference to that of RNA and protein.
The overall scheme of the protein synthesis machinery was outlined 40 years ago; however, structural studies of this huge and complicated system progressed very slowly until the final decade of the last century. In the 1990s, rapid progress was made in structural studies as a result of the many technical and methodical advances in gene engineering, biochemistry, electron microscopy, and x-ray crystallography. Thus, the crystal structures of prokaryotic ribosomes and all bacterial translation factors have been solved. Our group actively participated in those studies. We produced x-ray quality crystals of the 70S ribosome and 30S ribosomal subunit from Thermus thermophilus; we also solved the crystal structures of elongation factor G and ribosome recycling factor RRF and the crystal and NMR structures of more than 10 isolated ribosomal proteins and five complexes of ribosomal proteins with specific fragments of rRNAs.
Analysis of the RNA-binding sites in bacterial and archaeal ribosomal proteins L1 and of the corresponding L1-binding sites in archaeal rRNA and mRNA
One goal of our current research is to study the structures of regulatory complexes between ribosomal proteins and their mRNAs. We believe that comparative structural analysis of such regulatory complexes and complexes of the same proteins with ribosomal RNAs will shed light on the organization of recognition sites of protein and RNA molecules and enable us to understand the intermolecular interactions responsible for recognition.
Bacterial and archaeal ribosomal proteins L1 were shown to be able to regulate gene expression by binding to their own mRNAs, thereby acting as translational repressors. Binding sites for L1 proteins on 23S rRNA and on their mRNAs share a conserved structure. The proteins bind to the specific site on 23S rRNA with about one order of magnitude higher affinities than to their regulatory binding site on the mRNA. This difference fits the requirements of classical regulation of ribosomal synthesis (feedback inhibition) based on direct competition between the two binding sites. In 2003, our group published the structure of L1 in complex with a fragment of 23S rRNA. In 2005, we determined the structure of two different L1-mRNA complexes (for bacterial and archaeal L1 proteins). Comparative structural analysis of the ribosomal and regulatory complexes revealed the following: 1) the protein and the RNAs specifically interact through structurally invariant sites on their surfaces (recognition sites); 2) the difference in stability of ribosomal and regulatory complexes is determined by the contact area size; 3) in addition to the complementarity of interacting surfaces, recognition of the RNA target by the protein is determined by the atoms involved in conserved RNA-protein hydrogen bonds, which are inaccessible to the solvent, uncompensated losses of such hydrogen bonds leading to inhibition of complex formation; and 4) recognition of L1 proteins requires only domain I, but domain II plays a regulatory role by modulating the complex stability.
Knowledge of the high-resolution structures of L1-rRNA and L1-mRNA complexes has allowed us to design point mutations in the protein and its target RNAs so as to dissect interactions responsible for RNA-protein recognition and the stability of RNA-protein complexes.
Crystallization and structural studies of regulatory complexes of the Hfq protein
Bacterial protein Hfq regulates the expression of genes for many different proteins and is considered a global regulator of translation. The protein acts in complex with small regulatory RNAs. Hfq is a small (8 to 11 kDa), thermostable protein, which forms a hexameric ring. The ability of Hfq to associate with various RNAs, its binding to U-rich tracts of sRNAs, and the identification of an Sm1-like motif in the N-terminal portion of the protein allowed us to consider Hfq a bacterial equivalent of the eukaryotic and archaeal Sm/Lsm proteins, which are involved in RNA splicing and folding. Despite their very limited sequence homology, the structures of Hfq and Sm/Lsm monomers are virtually the same. However, the Hfq proteins and the archaeal Lsm2 protein form homo-hexamers, whereas other Sm and Lsm proteins function as homo- or hetero-heptamers. We are investigating the interactions between monomers that determine their mode of oligomerization. We recently solved the crystal structure of Hfq from Pseudomonas aeruginosa. Our comparative analysis of the known structures for proteins of the Sm/Lsm family revealed that the fragment of the Sm fold, which is responsible for oligomerization, is strongly conserved structurally. In the heptameric ring, three conserved hydrogen bonds, which are inaccessible to the solvent and are located between ၢ-strands of adjacent molecules, hold the monomers together, whereas in the hexameric rings of Hfq we observe an additional conserved inaccessible hydrogen bond between neighbor monomers. Mutational analysis can clarify the role of this hydrogen bond in stabilization of the hexameric ring.
There are several models for Hfq-RNA interactions. So far, only the structure of Hfq in complex with a model U-rich oligonucleotide has been determined. In this structure, the RNA is bound in a circular, unwound manner around the hole of the Hfq hexamer. To understand in detail mechanisms by which the protein might perform its function as modulator of RNA-RNA interactions, further structural studies of the Hfq-RNA complexes are needed. We also plan crystallization and structural studies of this protein in complex with small regulatory RNAs and fragments of the target mRNAs.
Crystallization and structural studies of translation initiation factor aIF2 and its functional complex aIF2*-GTP*-Met-tRNAiMet
In eukaryotes, the heterotrimeric translation initiation factor 2 (eIF2αβγ) delivers the initiator-tRNA (tRNAi) to the ribosome. The large γ subunit forms the core of the heterotrimer. The subunit interacts with both α and β subunits. In the trimer, the α and β subunits do not interact with each other. Phosphorylation of eIF2α, which occurs under a variety of conditions including viral infections, apoptosis, and so forth, prevents formation of the eIF2*-GTP*-Met-tRNAiMet complex and inhibits global protein synthesis. In this way, eIF2α appears to play a regulatory role, while eIF2γ binds to GTP and recognizes tRNAi. All known archaeal genomes encode polypeptides homologous to eIF2αβγ.
The structures of a/eIF2 (“a” denotes archaeal and “e” denotes eukaryotic) and its ternary complex with GTP and Met-tRNAiMet are of great interest. However, so far only the structures of the isolated subunits have been determined. Recently, our group began crystallization and structural studies of this important component of translation initiation in Archaea and Eukarya. So far, we have purified all three subunits of aIF2 from the hyperthermophilic archaeon Sulfolobus solfataricus on a preparative scale and have begun crystallization trials. We have crystallized the isolated γ subunit and determined its structure by the molecular replacement method. However, the main aim of this project is crystallization of the full trimeric factor and its ternary complex with GTP and Met-tRNAiMet. Determination of the structures of the heterotrimeric aIF2 and aIF2*-GTP*-Met-tRNAiMet complex should contribute toward a better understanding of the molecular details of translation initiation in Archaea and Eukarya. Ancillary projects include crystallization of the bacterial and archaeal ribosomal “L12 stalk” and the L10(L12)4 complex of ribosomal proteins in the free state and in complex with specific minimal fragments of 23S rRNA.
Our work is also supported by grants from the Russian Foundation for Basic Research, the Program of the Russian Academy of Sciences on Molecular and Cellular Biology, and the Program of the Russian Federation President for support of outstanding scientific schools.
Last updated March 2007
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