Structure and Function of the Ribosome Harry Noller, MCD Biology Ribosomes are RNA-based molecular machines that are responsible for synthesis of proteins. Researchers in the Noller laboratory were the first to solve the complete structure of a ribosome using X-ray crystallography. Besides the importance of protein synthesis to understanding the molecular basis of cellular function, research on ribosomes promises to improve the design of new antibiotics. Many of today's most effective anti-microbial drugs work by targeting bacterial ribosomes. As pathogenic bacteria continue to develop resistance to commonly used antibiotics, clarification of the structure and molecular mechanisms of bacterial ribosomes will be critical for the design of new drugs that will keep pace with rapidly evolving bacteria.

Ribosome Structure and Function

Ribosomes are complex molecular machines that are responsible for carrying out protein synthesis – translation of the genetic code. Their structures are highly conserved, and fundamentally similar in all organisms. A major question is, why do ribosomes contain such large amounts of RNA (50-60% of their mass)? It has gradually become clear that ribosomal RNA itself is centrally involved in translation, and may be a relic of the RNA World, a period of early molecular evolution when it is proposed that RNA carried out the genomic and catalytic roles of DNA and protein.

The Noller laboratory studies ribosome structure and function using a wide range of approaches, including X-ray crystallography, chemical probing methods, molecular genetics, comparative sequence analysis, fluorescence resonance energy transfer (FRET), including the use of single-molecule methods. The ultimate goal of these studies is to understand how the ribosome works at the molecular level: what are the moving parts of the machine, and how do they move in three dimensions to enable translation?

The three-dimensional structure of the complete 70S ribosome from the bacterium Thermus thermophilus at 3.7 Å resolution (Korostelev et al., 2006).

X-ray Crystal Structure of the Ribosome

We have recently solved the structure of the whole 70S ribosome from Thermus thermophilus at a resolution of 3.7 Angstroms. This has enabled us to describe all of the detailed molecular interactions of the ribosome, including ones involving the ribosomal RNAs, ribosomal proteins, tRNAs and mRNA. This provides a molecular basis for beginning to address the important functional questions. Our next goals for crystallography are to solve the structures of the ribosome trapped in different intermediate states of translation, including complexes containing elongation factors and other components.

Movement Inside the Translational Engine

On the basis of chemical footprinting results, we realized that the tRNAs move on the ribosome during the translocation step of protein synthesis in two steps: first they move at their acceptor CCA ends on the large ribosomal subunit, and then they move at their anticodon ends on the small subunit. We believe that both steps are catalyzed by the elongation factor EF-G, but the second step depends on GTP hydrolysis. We want to understand how the interactions between EF-G and the ribosomal machinery results in movement of the tRNAs and mRNA during translation. Clues from structural studies and from biochemical experiments implicate certain molecular features of ribosomal RNA (but also ribosomal proteins) in this movement. We are using chemical probing, FRET and mutational alteration of RNA and proteins to address these questions.

Functional Interactions of Translational Factors with the Ribosome

During protein synthesis, the ribosome interacts with initiation factors, elongation factors, release factors and ribosome recycling factor. Little is understood about their molecular interactions with the ribosome, and what they actually do to help the ribosome through the translational cycle. We are using chemical footprinting and directed hydroxyl radical probing to position the factors on the ribosome, and fluorescence methods to catch them in action. We are also attempting to crystallize complexes of the ribosome bound to the factors. Since most factor-catalyzed functions can be carried out by the ribosome itself under certain conditions, it is likely that these are all fundamentally mechanisms of the ribosome, whose rates and accuracy are increased by the factors.