Interaction of ribosomal proteins with their specific rRNA and mRNA binding sites: Structural features that modulate their affinity for each other

Research project P 14550 Interaction of ribosomal proteins with rRNA and mRNA Wolfgang PIENDL 26.6.2000 The regulation of synthesis of ribosomal proteins in Archaea, the third domain of life besides Bacteria and Eukarya, was studied in mesophilic and (hyper)thermophilc species of the methanogenic genus Methanococcus within the framework of a preceding FWF-funded project. In Methanococcus the Ll operon (encoding ribosomal proteins Ll, L10 and L12) is autoregulated by L I at the level of translation via a novel, not fully understood mechanism. L1 is a primary 23 S rRNA binding protein, that, when in excess, binds - with a tenfold lower affinity - to the regulatory binding site on its mRNA and thus inhibits translation of all three cistrons of the operon. In the course of this project we will try to pinpoint exactely the step of translation, at which methanoccal Ll inhibits its own synthesis. This step could be the formation of the 70S initiation complex or the binding of the aminoacyl-tRNA/EF-Tu/GTP ternary complex to the ribosome. The main aim of the new project is to study the interaction of ribosomal proteins with their specific rRNA and mRNA binding sites. Our work will concentrate on ribsomal protein L1, but also include S8 and the pentameric L10/L12(4) complex. The proteins from mesopilic; and thermophilic; Methanococcus species, which we use as a modell system, show a high degree of homology (- 80% identical amino acids), but the thermophilic proteins exhibit an at least tenfold higher affinity for their specific RNA binding sites as compared to their mesophilic counterparts. Bases and amino acids directly involved in the RNA-protein complex formation are identical in high- and low-affinity complexes. Our investigations will focus on the identification and, characterization of those structural features that determine the low- or high affinity binding character of the RNA binding proteins. Similarly, we will study the structural elements of the Ll -binding site on 23S rRNA and mRNA, that define them as a high or low affinity binding site. A combination of structural and functional data will provide insight by which stratagies RNA binding proteins and RNA binding sites for proteins are modulating their affinities for each other.

The most important results obtained in the frame of this project can be summarized as follows: Solution of the structure of the L1 protuberance of the ribosome. In collaboration with a Russian group the crystal structure of ribosomal protein L1 (from the Archaeon Sulfolobus acidocaldarius) in complex with its specific binding site on the 23S ribosomal RNA could be solved. Thus, a major gap in current structural models of the ribosome could be filled. Solution of the structure of the regulatory L1-mRNA complexes. In Bacteria and Archaea ribosomal protein L1 regulates its own synthesis (and that of the other ribosomal proteins encoded on the same transcriptional unit). When produced in excess, it binds to a structure on its own mRNA, which mimics the specific binding site on the 23S rRNA, with an 10fold lower affinity compared to that for the 23S rRNA. Thus it inhibits the translation of its own mRNA. In collaboration with the Russian group we solved the structure of the regulatory L1-mRNA complex. Comparison of the structures of the L1-mRNA with the L1-rRNA complex shows that fewer H-bonds involved in L1-mRNA interactions are responsible for the difference in affinity. Ribosomal proteins from (hyper)thermophilic organisms bind RNA with extremely high affinity. Primary rRNA binding ribosomal proteins S8 and L10 (as part of the L10/L124 complex) from thermophilic organisms (optimal growth temp. up to 75C) exhibit a 10-fold and proteins from hypertheromophiles (optimal growth temp above 75C) exhibit a 100-fold higher affinity for their specific rRNA binding sites than their closely related mesophilic counterparts. The stability of individual rRNA-protein complexes might modulate the stability of the ribosome, providing a maximum of thermostability and flexibility at the growth temperature of the organism. Our investigations will make a contribution to the general understanding of RNA-protein interactions, which play an important role not only in genesis and function of ribosomes, but also in many other biological processes, such as differentiation and regulation of gene expression.