Emory University School of Medicine
Ribosomal mRNA frameshifts mediated by RNA
The ribosome is a 2.5 million Dalton macromolecular machine responsible for the production of all proteins in every living organism. This enzyme is comprised of two asymmetric subunits containing protein and RNA that promote messenger RNA (mRNA)-directed translation of the genetic code. Understanding the detailed mechanism of ribosome function is important not only as a fundamental problem in biology, but also because many clinically relevant antibiotics target the ribosome.
A hallmark of the ribosome is its ability to strictly maintain the triplet reading frame as the mRNA, along with its correctly bound tRNA, moves through the three tRNA binding sites on the ribosome (A, P and E sites, figure above). Protein synthesis is an accurate process but errors do occur albeit rarely. One particular type of error that is extremely detrimental to a cell is mRNA frameshifting. This type of error is produced when the ribosome deviates from the normal genetic code and reads non-three-base codons on the mRNA. Therefore, the amino acid sequence downstream of the frameshift is nonsense and the protein is targeted for degradation.
In the case of retroviruses, reading of a non-three-base codon is an important mechanism for gene expression regulation and their survival. When these viruses infect cells they exploit ribosomal mRNA frame errors to express different proteins on overlapping genes upon demand from their limited genome. The causes of this frameshifting are a string of the same bases in the mRNA sequence in addition to complex, secondary structural motifs adopted by the mRNA. The molecular details of this mRNA-ribosome interaction are currently unknown. Our laboratory is interested in two distinct cases of frameshifts: ones caused by complex mRNA structures and others involved in suppression of mRNA frameshifts by tRNA mutations. Previously, we solved structures of the anticodon stem-loops from tRNAs bound to the ribosome that cause the mRNA to read four, rather than three, codon bases (see inset of figure above). These structures reveal how the ribosome is able to accommodate larger tRNA anticodon stem-loops by an unprecedented A-site codon-anticodon interaction. This work provides a snapshot of the interactions between tRNAs involved in frameshifting within the decoding center and sheds light on why some tRNAs, but not others, may promote +1 frameshifting. We anticipate that the interaction will be different depending upon the size of the ribosomal site the tRNA inhabits (i.e. A, P or E sites).
In eukaryotes, once properly spliced mRNAs are exported through the nuclear pore complex to the cytoplasm, the probability of whether they will be translation is directly correlated to the length of their polyA tail. In addition, mRNA secondary structural motifs also play important roles not only in determining translational fate but in all aspects of gene expression including splicing and export from the nucleus, translation initiation, and transport within in the cell for localized translation. The common theme among these diverse pathways is that mRNA structure plays the pivotal role in determining translational fate. My laboratory studies the molecular basis of how mRNAs are recognized by the ribosome and/or proteins involved in the repression or activation of protein synthesis, and how this specificity determines their biological function. At present, there is no molecular understanding of what drives the recruitment of RNA-binding proteins to specific mRNAs whether this recruitment mediates translational repression or activation. We are interested in how proteins recognize the 3’ UTR of different mRNAs that can either repress or activate translation and enzymes that process these mRNAs.