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.