mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding [preprint]

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation

Regulation of Ribosome Structural Dynamics by Antibiotics, Translation Factors and mRNA Secondary Structure

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2017.The structural dynamics of the ribosome underlie the mechanism by which information encoded in messenger RNAs is translated into a polypeptide chain. During protein synthesis, the small and large ribosome subunits rotate relative to each other while the L1 stalk, a mobile domain of large subunit, moves inward/outward relative to the core of the large subunit. Translation factors, antibiotics and structured RNAs are able to regulate or perturb translation by modulating ribosome structural dynamics. Furthermore, translation factors also undergo large structural rearrangements while interacting with the ribosome. Using single-molecule Förster resonance energy transfer (smFRET) to follow the structural rearrangements of the ribosome and translation factors, I have found (a) that the antibiotic, blastidicin S (BlaS), slows down intersubunit rotation, (b) the HIV and dnaX frameshift stimulating stemloops inhibit ribosome translocation by blocking tRNA binding to the A site of the ribosome, and (c) bacterial initiation factor 2 (IF2) positions ribosome subunits in a semi-rotated orientation during the subunit-joining step of translation initiation. Many small molecule antibiotics that target translation perturb ribosome structural dynamics. BlaS is a potent translation inhibitor in both eukaryotes and bacteria; however, the mechanism of BlaS action was not clear. I found that BlaS interacts with the P-site tRNA and inhibits spontaneous intersubunit rotation in bacterial ribosomes. Further studies by our collaborators from UMASS School of Medicine revealed that BlaS targets proteins synthesis via a unique mechanism hampering translation termination. During translation elongation, the ribosome moves along mRNA in a codon-by- codon manner and unwinds mRNA secondary structure. However, specific mRNA stemloops, such as the frameshift stimulating signal (FSS) from the E. coli dnaX gene, induce ribosome stalling and frameshifting. We sought to determine how FSS RNA stemloops stall ribosome translocation despite efficient helicase activity of the ribosome. Surprisingly, we found that the FSSs from HIV and dnaX mRNAs block tRNA binding to the A site of the ribosome, thereby inhibiting translocation mediated by the bacterial ribosome translocase, elongation factor-G. Our future studies will identify properties of mRNA secondary structure that inhibit A-site tRNA binding. Thus, our studies will provide fundamental insights into the regulation of translation elongation by mRNA structure and the mechanism of programmed ribosome frameshifting. We also extended our studies beyond translation elongation to elucidate the molecular mechanism of the subunit-joining step of translation initiation in bacteria. Initiation of protein synthesis is the key regulatory step of translation. In bacteria, translation initiation is controlled by initiation factors (IFs) 1, 2 and 3, which ensure selection of the initiator tRNA and promote joining of the large and small ribosomal subunits. Using smFRET, we find that IF2, a translational GTPase, transiently stabilizes the ribosome in a distinct conformation where the ribosomal subunits are in a semi- rotated orientation and the L1 stalk is in a half-closed position (3). Our results suggest that positioning subunits in a semi-rotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding

Interplay between Inter-Subunit Rotation of the Ribosome and Binding of Translational GTPases

Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP·fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon–anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G·GDP. Unexpectedly, translocation with EF-G·GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G·GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP·fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G·GTP or EF-G·GDPNP

Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome

The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-A crystal structure of BlaS bound to a 70StRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3\u27 terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation

Following movement of the L1 stalk between three functional states in single ribosomes

The L1 stalk is a mobile domain of the large ribosomal subunit E site that interacts with the elbow of deacylated tRNA during protein synthesis. Here, by using single-molecule FRET, we follow the real-time dynamics of the L1 stalk and observe its movement relative to the body of the large subunit between at least 3 distinct conformational states: open, half-closed, and fully closed. Pretranslocation ribosomes undergo spontaneous fluctuations between the open and fully closed states. In contrast, posttranslocation ribosomes containing peptidyl-tRNA and deacylated tRNA in the classical P/P and E/E states, respectively, are fixed in the half-closed conformation. In ribosomes with a vacant E site, the L1 stalk is observed either in the fully closed or fully open conformation. Several lines of evidence show that the L1 stalk can move independently of intersubunit rotation. Our findings support a model in which the mobility of the L1 stalk facilitates binding, movement, and release of deacylated tRNA by remodeling the structure of the 50S subunit E site between 3 distinct conformations, corresponding to the E/E vacant, P/E hybrid, and classical states