A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed −1 ribosomal frameshifting (−1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine–Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA^Lys in the ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as −1PRF.The ribosome is the molecular machine that synthesizes proteins by translating messenger RNAs (mRNAs); each sequence of 3 nt, 1 codon, characterizes 1 aa (1–3). Failure to maintain frame during translation occurs with a low error of 10⁻⁵ (4); however, frameshifting with high efficiency (>10⁻²) is often programmed into many mRNAs to express two or more proteins from a single mRNA (5, 6). Many RNA viruses, including HIV-1, use programmed frameshifting to produce their vital proteins at a precise ratio (7, 8). The common −1 programmed ribosomal frameshifting (−1PRF) signals are a heptanucleotide slippery sequence (X XXY YYZ, underlining denotes the zero-frame) and a downstream stimulatory secondary structure such as a stem loop or a pseudoknot. Frameshifting that takes place on the slippery sequence results in minimal base pair mismatches. Prokaryotic systems have an additional stimulatory signal, an upstream, internal Shine–Dalgarno (SD) sequence (9). The dnaX gene of Escherichia coli has the three −1PRF signals; an SD sequence, an A AAA AAG slippery sequence, and a downstream stem loop (9–12). Highly efficient (50–80%) −1PRF during translation of the mRNA results in production of the γ DNA-polymerase subunit in the −1 frame and the τ DNA-polymerase subunit in the 0 frame (10).The −1PRF signals are spaced so that the slippery sequence is positioned within the ribosomal peptidyl-tRNA–binding (P) site and aminoacyl-tRNA–binding (A) site, whereas the downstream secondary structure is positioned at the ribosomal mRNA entry channel (Fig. 1) (5–8, 13). The upstream SD sequence base pairs with 16S ribosomal RNA (rRNA) near the ribosomal tRNA exit (E) site (Fig. 1) (9). Both the SD sequence and the downstream secondary structure can cause pausing during translation (14–19). However, frameshifting efficiency is not strictly related to the pausing extent (15, 17), and it is not proportional to the thermodynamic or mechanical stabilities of the secondary structures (7, 20). Nonetheless, it does correlate with the thermodynamic stability of the first 3–4 bp of the downstream secondary structure (21), and with the conformational plasticity of this structure (7, 20). However, a mechanism by which the stimulatory secondary structure promotes efficient frameshifitng has not emerged yet.Open in a separate windowFig. 1.A programmed −1 FSmRNA construct and a schematic drawing of a ribosomal complex translating the slippery sequence. FSmRNA contains three −1PRF signals from the dnaX gene in E. coli; an SD sequence, a slippery sequence, and a downstream stem loop. ΔSL mRNA has the same sequence as FSmRNA except with the stem loop (red box) deleted. Start and stop codons are highlighted in blue. Corresponding polypeptide sequences are shown below the mRNA. A schematic drawing of the POST-(Cy5)K₁ complex shows the 50S and 30S subunits in blue and purple rectangles, respectively. The L1 stalk in the small blue rectangle is labeled with Cy3. The ribosomal complex contains fMVK-(Cy5)tRNA^Lys in the P site, where the slippery sequence is being displayed. The upstream SD sequence forms base pairs with 16S rRNA and the downstream stem loop presents at the mRNA entry channel in the 30S subunit. The orange oval denotes the biotin on a DNA primer annealed to the 5′ end of the mRNA for immobilization.A translational elongation cycle starts with selecting a correct aminoacyl-tRNA in the A site via conformational changes of the posttranslocation (POST) ribosomal complex that are triggered upon binding an EF-Tu(GTP)⋅aminoacyl-tRNA ternary complex (TC) (1). Once peptidyl transfer takes place, the resulting pretranslocation (PRE) ribosomal complex undergoes large-scale conformational changes that facilitate translocation of the tRNAs from the P and A sites into the E and P sites, simultaneously advancing the ribosome along the mRNA by 3 nt (22). In the first step of translocation, the acceptor stems of the tRNAs are repositioned within the large ribosomal (50S, in prokaryotes) subunit to move the tRNAs from their classical (P/P, A/A) state to their hybrid (P/E, A/P) states, where X and Y in the X/Y notation refer to the position of the anticodon stem loop (ASL) of the tRNA in the small ribosomal (30S, in prokaryotes) subunit and the position of the acceptor stem of the tRNA in the 50S subunit, respectively. Hybrid state (H) formation is accompanied by rotation of the 30S subunit relative to the 50S subunit (23, 24) and a closure of the L1 stalk of the 50S subunit such that it forms a direct contact with the P/E hybrid tRNA (23–25), a global conformation of the PRE complex that we refer to as “global state 2” (25). Global state 1, in contrast, contains classical state (C) tRNAs, nonrotated subunits, and an open L1 stalk (25). Single-molecule fluorescence resonance energy transfer (smFRET) studies of this step of translocation have shown that the H state forms spontaneously upon peptidyl transfer and that, in the absence of an elongation factor-G (EF-G), the H state exists in a dynamic equilibrium with the C state (25–27). Translocation is completed by movement of the ASLs of the tRNAs and the mRNA in the 30S subunit. This step, which comprises the rate-limiting step for the overall process of translocation, requires unlocking of the PRE complex, a conformational change that is thought to involve swiveling of the head domain of the 30S subunit (28, 29) and that is catalyzed by EF-G (30). smFRET and structural studies suggest that the L1 stalk–P/E hybrid tRNA interaction that is established during the first step of translocation is preserved throughout the second step of translocation and is essential for guiding the translocation of the P/E hybrid tRNA into the E site (25, 31, 32).Here, we report an smFRET study of the dynamics of ribosomal complexes programmed with the −1PRF mRNA of the E. coli dnaX gene. We used a FRET pair composed of a Cy3-labeled L1 stalk [L1(Cy3)-stalk] and a Cy5-labeled P-site tRNA^Lys [(Cy5)tRNA^Lys] on the first lysine codon in the slippery sequence. As previously demonstrated (25), this FRET pair enabled us to monitor transitions of ribosomal complexes between C and H states and the subsequent release of the translocated (Cy5)tRNA^Lys from the E site, along one round of the translational elongation cycle. Two mRNA constructs, one containing the downstream stem loop and one lacking it, were compared to study the effect of the secondary structure on the dynamics and translocation of the ribosomal complexes. Our results show that the downstream stem loop changes the dynamics of the PRE ribosomal complexes and disturbs the translocation process. We propose that frameshifting is one of the favorable paths that the ribosome can adopt during the futile EF-G–driven translocation attempts from the H state.     

Regulators of G protein signaling RGS7 and RGS11 determine the onset of the light response in ON bipolar neurons

The time course of signaling via heterotrimeric G proteins is controlled through their activation by G-protein coupled receptors and deactivation through the action of GTPase accelerating proteins (GAPs). Here we identify RGS7 and RGS11 as the key GAPs in the mGluR6 pathway of retinal rod ON bipolar cells that set the sensitivity and time course of light-evoked responses. We showed using electroretinography and single cell recordings that the elimination of RGS7 did not influence dark-adapted light-evoked responses, but the concurrent elimination of RGS11 severely reduced their magnitude and dramatically slowed the onset of the response. In RGS7/RGS11 double-knockout mice, light-evoked responses in rod ON bipolar cells were only observed during persistent activation of rod photoreceptors that saturate rods. These observations are consistent with persistently high G-protein activity in rod ON bipolar cell dendrites caused by the absence of the dominant GAP, biasing TRPM1 channels to the closed state.     

Modulation of regulatory T-cell activity in combination with interleukin-12 increases hepatic tolerogenicity in woodchucks with chronic hepatitis B

Regulatory T cells (Treg) play a critical role in the modulation of immune responses to viral antigens in chronic viral hepatitis. Woodchucks (Marmota monax) infected with the woodchuck hepatitis virus (WHV) represent the best animal model for chronic hepatitis B virus (HBV) infection. Examination of intrahepatic and peripheral Treg in uninfected and WHV chronically infected woodchucks showed a significant increase of intrahepatic Treg numbers in chronically infected animals, whereas no differences were found in peripheral blood. In agreement with these data, higher expression levels of Forkhead box P3 (Foxp3), interleukin (IL)-10, transforming growth factor beta (TGF-β) were detected in the liver of chronic WHV carriers in comparison to uninfected animals. Furthermore, treatment of WHV-infected animals with an adenovirus encoding IL-12 failed to reduce viral load, a finding that was associated with lymphocyte unresponsiveness to IL-12 stimulation in vitro. We observed that TGF-β and Treg play a major role in the lack of lymphocyte response to IL-12 stimulation, as TGF-β inhibition and Treg depletion allowed recovery of T-cell responsiveness to this cytokine. Based on these results, woodchucks were treated with IL-12 in combination with a TGF-β inhibitory peptide or Treg depletion. However, no antiviral effect was achieved and, instead, an enhancement of the intrahepatic tolerogenic environment was observed. CONCLUSION: Our data show that TGF-β inhibition or Treg depletion had no added benefit over IL-12 therapy in chronic WHV infection. IL-12 immunostimulation induces a strong immunosuppressive reaction in the liver of chronic WHV carriers that counteracts the antiviral effect of the treatment.