Structural and functional analysis of ribosome initiation and ribosomal frameshifting.

Lead Research Organisation: University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics


Proteins are polymers of amino acids. The order of the amino acids is governed by the genetic code in the genes of the cell's DNA. In protein synthesis, a gene is first copied into messenger RNA (mRNA) - a single-strand copy of the DNA which retains the code - and then taken to the ribosome. Ribosomes are protein synthesising machines some 25-35nm in diameter and made up of two subunits, one large, one small. The mRNA is loaded linearly between the subunits, whereupon adaptor molecules called transfer RNAs (tRNAs) arrive to decode the mRNA. Each transfer RNA has an amino acid at one end, and at the other, a region which recognises a specific run of three consecutive nucleotides in the mRNA code, a 'triplet'. During protein synthesis, the ribosome moves along the mRNA triplet by triplet and at each step, a tRNA with the appropriate recognition sequence is recruited and the ribosome transfers the tRNA's amino acid to the growing chain of amino acids, the polypeptide. This project will study three aspects of protein synthesis. The first is how mRNA is recruited to the ribosome and fed between the subunits. In mammals, the mRNA interacts with the isolated small subunit of the ribosome in complex with a host of other proteins termed initiation factors. This initiation complex moves along the mRNA, scanning the sequence until the specific triplet that signifies the start of protein synthesis is identified. At this point, the large subunit joins and amino acid polymerisation begins. We wish to characterise in detail the structure of this scanning complex (termed the 48S complex), by purifying initiation complexes paused in the act of scanning along the mRNA, or at the initiation triplet, and study them using microscopy. Our microscope uses electrons rather than light, and is extremely powerful because the short wavelength of electrons allows the visualisation of fine molecular detail. The samples are also frozen at -180C, which keeps them in a natural and stable state, and so the technique is known as 'cryo-electron microscopy' (cryo-EM). The images we obtain will show us the structure of the 48S complex and tell us something of how the various initiation factors allow the small subunit to scan along the mRNA and to start protein synthesis. The second aspect is elongation, when amino acids are added, via tRNAs, to the growing chain. Inside the ribosome are multiple binding sites for tRNAs. A tRNA comes in at one site, binds to its complementary mRNA triplet and transfers its amino acid to the growing chain, moves to an adjacent site to make space for the next tRNA, and finally leaves the ribosome via an exit site. The movement of tRNAs has been hard to analyse at a molecular level. We have identified an mRNA signal termed a pseudoknot which blocks the progress of the ribosome along the mRNA and stalls the ribosome at the point when the tRNAs are moving within the ribosome. We will use cryo-EM to study the structure of ribosomes stalled at a pseudoknot to elucidate the molecular details of tRNA movement. Pseudoknots can also cause the ribosome to change reading frame, that is, to shift from reading one set of triplets (one frame) to reading an overlapping set (another frame), and this leads to a completely new amino acid sequence in the protein. Cryo-EM studies of pseudoknot-stalled ribosomes may thus be informative as to how the ribosome maintains the correct reading frame. The final aspect of the project concerns the structure of ribosomes prepared from mammalian cells. Because the ribosome plays a major role in controlling the rate of protein synthesis, and even which proteins are made, in different cell states the ribosome becomes modified in a variety of ways that affect which proteins are made and how rapidly. By determining structures for ribosomes from cells grown under known conditions we will make the first moves in showing the relationship between ribosome mechanism and regulatory processes in living cells.

Technical Summary

Protein synthesis requires that mRNAs be recruited to the ribosome and processed through it, the genetic code being read into an amino acid sequence. mRNA recruitment involves a series of eukaryotic initiation factors (eIFs) that bind to and modify the architecture of the small ribosomal subunit. Two key complexes are formed during this process: the 43S complex in which the 40S small subunit is bound with several protein initiation factors (eIFs) that open its structure up to bind mRNA, and the mRNA-bound form which has additional eIFs bound and is known as the 48S complex. We aim to purify 48S complexes from mammalian in vitro translation reactions derived from rabbit and human cells and to determine its structure by cryo-electron microscopy (cryo-EM). This will allow us to show how the host of eIFs facilitate mRNA recruitment and scanning to find the AUG start codon. In a parallel project, we will continue our previous work on ribosomes at the polypeptide elongation phase of protein synthesis. The translocation of tRNAs through the ribosomal intersubunit space maintains the triplet codon reading frame. Specific signals in the mRNA itself can cause frameshifting, either -1 or +1, to generate a different protein sequence. Current data indicate a molecular spring-and-ratchet mechanism for translocation of tRNA, confounded in the presence of frameshift-stimulating secondary structure in the mRNA, which generates the reading frame change. We will study pseudoknot and stem-loop folded mRNAs of known structure in the context of the translating ribosome to determine in greater detail how they cause the frameshift to occur. A further study involving 80S ribosomes will look at how protein synthesis is regulated in the cell during the elongation phase of synthesis. This will focus on the modifications accompanying changes in cell growth rate, and how this is reflected in the conformation of the ribosome and its binding by regulatory proteins and other ligands.
Description -That there are two different hybrid states between the A and P sites of the ribosome (A/P' and A/P") that have differing tRNA conformations.

-That frameshifting analysis has shed light on the process of regular tRNA translocation.

-That RNA interference -1 or -2 nucleotides on mRNA is dependent on the spacing between the slippery site and the bound RNA.

-That modification of pseudoknots by the inclusion of additional loops changes the frameshifting complex assembly (to be published).

-That -1 frameshifting caused by a stem-loop in HIV mRNA is similar in mechanism to that caused by a coronavirus mRNA pseudoknot (to be published).

-That the antibiotic cycloheximide causes ribosomes to be frozen with tRNAs in hybrid states A/P and P/E (to be published).

-That the mammalian 48S initiation complex involves reconfiguration of the small subunit (to be published).
Exploitation Route Has the potential to be applied as a therapeutic target in disease caused by viruses such as HIV and coronaviruses.
Sectors Education

Description They have led to several papers from other groups testing the mechanical model we developed from our work.
First Year Of Impact 2007
Sector Education
Impact Types Cultural

Description Association of Resources for Biophysical Research in Europe
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact We are implementing Europe-wide projects for quality control of biophysical methods and benchmarking of new technologies. Our role builds directly on biophysical and structural biology studies carried out in the lab over several years. It has been strengthened by additional funding which began since our involvement with ARBRE.
Description Molecular Biophysics in Europe (MOBIEU)
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact MOBIEU is a COST Action which will enable the improvement of biophysical methods development across Europe and with international impact. I am one of two UK members of its Management Committee, nominated through the Department for Business, Information and Skills.
Title WellMAP 
Description A new computer program allowing rapid manipulation and assessment of 3D electron density maps. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact It has greatly improved the efficiency of our data analysis. 
Title WellMAP 
Description A newly encoded set of operations including novel computational tools for 3D map quality assessment and manipulation. 
Type Of Material Computer model/algorithm 
Provided To Others? No  
Impact It has greatly increased the efficiency of our data analysis. 
Description The mechanical basis of +1 frameshifting 
Organisation University Paris Sud
Country France 
Sector Academic/University 
PI Contribution A similar study to that performed on the funded project concerning -1 frameshifting, but studying a mammalian +1 frameshift. The structures of ribosomes from a +1 frameshift system show that the mechanisms for -1 and +1 frameshifts are distinct.
Collaborator Contribution They provided stamples for imaging analysis and also performed validating experiments on the samples used. This funding enabled this collaboration to begin - and a publication is still pending.
Impact This is a multidisciplinary project - biophysics and structural biology combined with cellular and molecular biology. A paper will be submitted shortly.
Start Year 2011
Description The mechanical basis of -1 frameshifting 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We collected cryo-EM image data and determined 3D reconstructions of the imaged ribosomal complexes.
Collaborator Contribution Dr Ian Brierley's team in Cambridge provided -1 frameshifting complexes for our analysis by structural methods. This funding strengthened our work. Publications from it are still pending.
Impact This is multidisplinary - we are biophysicists and structural biologists, Dr Brierley is a virologist and molecular biologist. Outputs are captured in our several papers.
Title WellMAP 
Description WellMAP encodes a set of operations some of which are standard in EM data analysis (eg Fourier shell correlation) and some of which are novel (the Fourier plate correlation) while still others are known elsewhere but were independently coded (B factor calculation and use for data filtration). 
Type Of Technology Software 
Year Produced 2011 
Open Source License? Yes  
Impact We have found the software has greatly improved our efficiency of data analysis. 
Description School visit (to Wellcome Trust Centre) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The talks revealed the value of a structural understanding of biological processes.

There seemed a heightened awareness of the value of our work.
Year(s) Of Engagement Activity 2012,2013