New Insights Into Protein Synthesis And Hepatitis C Infections

Biochemist Jennifer Doudna and biophysicist Eva Nogales, both of whom hold joint appointments with the Lawrence Berkeley National Laboratory (Berkeley Lab), the University of California at Berkeley, and the Howard Hughes Medical Institute (HHMI), led a study in which cryo electron microscopy (cryo-EM) was used to create a 3-D model of the protein complex called eukaryotic translation initiation factor 3 (eIF3). The model showed that the eIF3 protein complex employs the same structural mechanics in the loading of either human or viral RNA to ribosomes, the complex machinery in living cells responsible for protein synthesis.

“This is the first insight into how the initiation mechanisms of protein synthesis work specifically for humans, and a step towards understanding at the molecular level what happens when a viral infection occurs,” said Doudna, a member of Berkeley Lab’s Physical Biosciences Division. “A better understanding of these mechanisms could open the door to new and improved therapies for viral infections.”

Said Nogales, also a member of Berkeley Lab’s Physical Biosciences Division, “Using cryo-EM, we can reconstruct images of the entire protein ensemble to study the molecular machinery behind the protein synthesis process. We now have the tools to see how the many different parts of the molecular machinery come together.”

The results of this study are in the December 2, 2005 issue of the journal Science, in a paper entitled
Structural Roles for Human Translation Factor eIF3 in Initiation of Protein Synthesis. Co-authoring the paper with Doudna and Nogales were Bunpote Siridechadilok and Christopher Fraser of UC Berkeley, and Richard Hall of Berkeley Lab.

Proteins, the curiously-shaped macromolecules that serve as the basic construction material of all living cells, and also initiate and control nearly all cell chemistry, are assembled out of amino acids according to the instructions contained within the genes. These genetic instructions are carried from the DNA inside a cell’s nucleus out into the cell’s cytoplasm via messenger RNA (mRNA). There the information will be translated to a sequence of amino acids via the ribosome, an ancient organelle so highly conserved by evolution that its core components are pretty much the same for all forms of life.

Protein synthesis in mammalian cells begins with the loading of mRNA onto the small ribosome subunit, 40S, which is, in part, one of the responsibilities of the eIF3 complex. The eIF3 complex also interacts with other translation elements that bind at the start of the mRNA, prevents premature joining of the 40S and 60S ribosomal subunits, and helps assemble active ribosomes. Until now, the structural basis for eIF3’s multiple activities has been unknown.

At a resolution of 30 angstroms, the cryo-EM reconstructions of Doudna and Nogales and their collaborators show eIF3 to be a particle consisting of five lobes - analogous to a head, and a pair of arms and legs. The study shows that the left arm of the eIF3 complex binds to the eukaryotic protein complex that recognizes the methylated guanosine cap at the 5’-end of the eukaryotic mRNAs (mRNA consists of a coding region sandwiched between a 5’-end and a 3’-end). By drawing the mRNA’s 5’-end cap through the ribosome entry site and towards the exit, eIF3 ensures the mRNA is properly positioned for its genetic code to be translated.

Acting like a molecular wrestler, eIF3 will also wrap its arms and legs around a structural element of RNA for the hepatitis C virus (HVC), known as the internal ribosome entry site (IRES), and pin it to the exit site of the 40S ribosome subunit. The IRES leaves through the left arm of the eIF3 complex at the same location where interaction with the human mRNA cap-binding complex takes place.

“This might explain the amazing ability of the HVC IRES to hijack the human ribosome and its associated translation factors,” said Doudna.

Said Nogales, “The position of eIF3 in our models also provides a plausible explanation for its role in preventing premature joining of the 40S and 60S ribosome subunits.”

Doudna and members of her research group are now working to improve the resolution of these models from 30 angstroms to about 10 angstroms. This would allow them to see secondary protein structures which would give them a better understanding of the chemistry behind eIF3’s structural mechanics.

Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.