The Noller Lab
The Noller lab studies the structure and function of the ribosome, the cellular ribonucleoprotein complex responsible for translation of the genetic code, and synthesis of proteins in all organisms. Ribosomes are highly conserved, with functional sites that are nearly identical across the phylogenetic spectrum. One of our main contributions has been to show the functional importance of ribosomal RNA (rRNA) in protein synthesis. We have provided evidence for the participation of rRNA in peptide bond formation, tRNA binding, interaction of ribosomes with translational initiation and elongation factors, binding of antibiotics and ribosomal subunit association. The first sequences of the large 16S and 23S rRNAs were obtained in our lab, and their secondary structures deduced by comparative sequence analysis, in collaboration with Woese. We have developed methods for chemical footprinting of RNA, and have used it to map the rRNA binding sites of the 30S subunit ribosomal proteins, the A-, P- and E-site tRNAs, translation factors and antibiotics. Chemical footprinting of ribosomes during steps of in vitro translation led to the hybrid states model for translocation. Development of directed hydroxyl radical probing methods allowed us to position ribosomal proteins, translation factors and tRNAs in three dimensions relative to structural features of rRNA. In recent yeasrs, we have used X-ray crystallography to solve structures of functional complexes of the complete ribosome containing mRNA and two or three tRNAs.
At present, we are trying to understand how the three-dimensional structure of the ribosome defines its functional properties. It is clear that the ribosome is a molecular machine. Among the key questions are, how does the ribosome move, and what are its moving parts? To this end, we are developing methods to study ribosome movement at the molecular level in real time. In collaboration with the Clegg and Ha groups (Illinois), we are studying ribosomal movement with fluorescence resonance energy transfer (FRET), using both bulk and single-molecule approaches. In collaboration with the Tinoco and Bustamante laboratories (UC Berkeley) we are using optical tweezers to measure molecular forces that occur during ribosome movement. The most dramatic dynamic event of protein synthesis is the tranlocation step, in which tRNAs and mRNA are moved rapidly and accurately through the ribosome over distances of 20 to 70 , catalyzed by elongation factor EF-G and GTP. We discovered that the small molecular weight antibiotic sparsomycin can catalyze translocation in the absence of EF-G and GTP. This surprising results shows that translocation is a property of the ribosome itself, and not of EF-G, and that the source of energy for translocation comes not from GTP, but from peptide bond formation. Recently, we tested the "ratchet" model for translocation based on cryo-EM reconstructions in the Frank laboratory, in which movement of mRNA and tRNa during protein synthesis were proposed to be driven by intersubunit rotational movement. Formation of a disulfide bridge across the subunit interface specifically abolished translocation, showing that intersubunit movement is essential. In bulk and single-molecule FRET studies, we have been able to observe this intersubunit movement as it occurs in real time.
We are also using genetics and mutational analysis of both ribosomal proteins and rRNA to study ribosomal function, using methods that allow us to isolate pure populations of mutant ribosomes for in vitro functional analysis. We are basing our studies primarily on questions that are prompted by inspection of the X-ray structure of the ribosome. For example, to test the idea that ribosomal function is based on RNA, rather than protein, we have deleted the tails of proteins S9 and S13, which are the only protein moieties in the 30S subunit P site. Cells whose ribosomes contain only rRNA in their 30S P sites show only moderate decreases in growth rate, in keeping with the 'RNA World' model: that the original ribosomes contained only RNA in their structures. In addition, directed mutagenesis of key regions of the rRNA is being carried out to test models for how rRNA functions in protein synthesis.
Finally, we are continuing to use X-ray crystallography to
study ribosome structure and function. During the past year,
we have extended the resolution of our 70S ribosome crystal
structure to 3.7, revealing details of the interactions
between mRNA, tRNA and the ribosome. We are continuing to
crystallize new functional complexes of ribosomes in
intermediate states of translation, and with bound
translational factors, with the ultimate goal of obtaining
an atomic, three-dimensional movie of translation.