Frameshifting Activity Research Articles

Sarbecovirus programmed ribosome frameshift RNA element folding studied by NMR spectroscopy and comparative analyses.

The programmed ribosomal frameshift (PRF) region is found in the RNA genome of all coronaviruses and shifts the ribosome reading frame through formation of a three-stem pseudoknot structure, allowing the translation of essential viral proteins. Using NMR spectroscopy, comparative sequence analyses and functional assays we show that, in the absence of the ribosome, a 123-nucleotide sequence encompassing the PRF element of SARS-CoV-2 adopts a well-defined two-stem loop structure that is conserved in all SARS-like coronaviruses. In this conformation, the attenuator hairpin and slippery site nucleotides are exposed in the first stem-loop and two pseudoknot stems are present in the second stem-loop, separated by an 8-nucleotide bulge. Formation of the third pseudoknot stem depends on pairing between bulge nucleotides and base-paired nucleotides of the upstream stem-loop, as shown by a PRF construct where residues of the upstream stem were removed, which formed the pseudoknot structure and had increased frameshifting activity in a dual-luciferase assay. The base-pair switch driving PRF pseudoknot folding was found to be conserved in several human non-SARS coronaviruses. The collective results suggest that the frameshifting pseudoknot structure of these viruses only forms transiently in the presence of the translating ribosome. These findings clarify the frameshifting mechanism in coronaviruses and can have a beneficial impact on antiviral drug discovery.

Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit.

The −1 programmed ribosomal frameshifting (−1 PRF) has been explored as a gene regulatory circuit for synthetic biology applications. The −1 PRF usually uses an RNA pseudoknot structure as the frameshifting stimulator. Finding a ligand-responsive pseudoknot with efficient −1 PRF activity is time consuming and is becoming a bottleneck for its development. Inserting a guanine to guanine (GG)–mismatch pair in the 5′-stem of a small frameshifting pseudoknot could attenuate −1 PRF activity by reducing stem stability. Thus, a ligand-responsive frameshifting pseudoknot can be built using GG-mismatch–targeting small molecules to restore stem stability. Here, a pseudoknot requiring stem–loop tertiary interactions for potent frameshifting activity was used as the engineering template. This considerably amplified the effect of mismatch destabilization, and led to creation of a mammalian −1 PRF riboswitch module capable of mediating premature translation termination as a synthetic regulatory mode. Application of the synthetic circuit allowed ligand-dependent ATF6N mimic formation for the activation of protein folding–related genes involved in the unfolded protein response without an ER-stress inducing agent. With the availability of mismatch-targeting molecules, the tailored module thus paves the way for various mismatch plug-ins to streamline highly efficient orthogonal ligand-dependent −1 PRF stimulator development in the synthetic biology toolbox.

MRNA-Mediated Duplexes Play Dual Roles in the Regulation of Bidirectional Ribosomal Frameshifting.

In contrast to −1 programmed ribosomal frameshifting (PRF) stimulation by an RNA pseudoknot downstream of frameshifting sites, a refolding upstream RNA hairpin juxtaposing the frameshifting sites attenuates −1 PRF in human cells and stimulates +1 frameshifting in yeast. This eukaryotic functional mimicry of the internal Shine-Dalgarno (SD) sequence-mediated duplex was confirmed directly in the 70S translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between 70S and 80S ribosomes. Unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of +1 frameshifting and stimulation of a non-canonical −1 frameshifting within the +1 frameshift-prone CUUUGA frameshifting site in the absence of release factor 2 (RF2) in vitro. However, the −1 frameshifting activity of the downstream pseudoknot is not coupled with its +1 frameshifting attenuation ability. Similarly, the +1 frameshifting activity of the upstream hairpin is not required for its −1 frameshifting attenuation function Thus, each of the mRNA duplexes flanking the two ends of a ribosomal mRNA-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other.

Introducing a class of standardized and interchangeable parts utilizing programmed ribosomal frameshifts for synthetic biology applications.

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.

Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins

RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3′ direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) −1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate−1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for−1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing−1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study’s findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.

Simulated and Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

Mechanical unfolding of −1 frameshift signals such as RNA pseudoknots have aimed to test the hypothesis that the stability of the pseudoknot (PK) is directly correlated to the frameshifting efficiency. Here we report unfolding of the Beet Western Yellow Virus (BWYV) PK by optical tweezers complemented by computer simulations using steered molecular dynamics (SMD). Three BWYV PK scenarios were studied: the wild-type PK in the presence and absence of Mg2+, and mutations of nucleic base C8 known to completely abolish −1 frameshifting by disrupting pseudoknot stability at the core of its structure. Despite significant differences in loading rates, we found the experimental and computational results to be remarkably consistent.The SMD simulations provide a detailed sequence of molecular unfolding events that can be assigned to the force-extension profiles obtained with the optical tweezers. In the absence of Mg2+, stretching of the PK using the optical tweezers does not result in the observation of any unfolding transitions, which is consistent with the SMD simulation that demonstrates the essential role of Mg2+ for the formation of a very strong salt bridge between G4, C5, G16, and C17 nucleotides. The C8 mutants, like wild-type unfolding in the absence of Mg2+, unfold readily and at low force, consistent with the absence of any −1 frameshifting activity for these mutants.

Mammalian Gene PEG10 Expresses Two Reading Frames by High Efficiency –1 Frameshifting in Embryonic-associated Tissues

Paternally expressed gene 10 (PEG10) is a mammalian gene that is essential for embryonic development in mice. The gene contains two overlapping open reading frames (ORF1 and ORF2) and is derived from a retroelement that acquired a cellular function. It is not known if both reading frames are required for PEG10 function. Synthesis of ORF2 would be possible only if programmed -1 frameshifting occurred during ORF1 translation. In this study the frameshifting activity of PEG10 was analyzed in vivo, and a potential role for ORF2 was investigated. Phylogenetic analysis demonstrated that PEG10 is highly conserved in therian mammals, with all species retaining the elements necessary for frameshifting as well as functional motifs in each ORF. The frameshift site of PEG10 was highly active in cultured cells and produced the ORF1-2 protein. In mice, endogenous ORF1 and an ORF1-2 frameshift protein were detected in the developing placenta and amniotic membrane from 9.5 days post-coitus through to term with a very high frameshift efficiency (>60%). Mutagenesis of the active site motif of a putative protease within ORF2 showed that this enzyme is active and participates in post-translational processing of PEG10 ORF1-2. Both PEG10 proteins were also detected in first trimester human placenta. By contrast, neither protein expression nor frameshifting was detected in adult mouse tissues. These studies imply that the ORF1-2 protein, synthesized utilizing the most efficient -1 frameshift mechanism yet documented in vivo, will have an essential function that is intrinsic to the importance of PEG10 in mammals.

Mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses.

Expression of the putative replicase of potato leafroll virus (PLRV) is regulated by -1 ribosomal frameshifting in which a primary viral transcript has two overlapping open reading frames (ORFs). A region of 39 nt at the junction of the two ORFs is essential for frameshifting to occur. It has been shown to harbor two signals, one active on the level of the primary structure, termed the slippery sequence, and one component that forms a secondary or tertiary level structure, described as either a pseudoknot or a stem-loop motif. We have performed extensive site-directed mutagenesis of the frameshifting region and analyzed individual mutants for their ability to promote -1 frameshifting in vitro. Detailed comparison of our results with analogous mutants in the frameshifting region of the evolutionarily related beet western yellow virus, for which a crystal structure is available, unequivocally argues for the pseudoknot to be the structural motif necessary for the frameshifting function in PLRV transcripts. Mutations in PLRV that affect putative pseudoknot-specific tertiary-base interactions drastically affect frameshifting activity. In addition, a specific deletion mutant was identified that displayed PLRV wild-type frameshifting activity with only 22 nt available for pseudoknot formation.

Slip-Sliding the Frame: Programmed −1 Frameshifting on Eukaryotic Transcripts

The basic mechanisms of mRNA translation are ubiquitous among all organisms, in that the accurate decoding of triplet codon sequences programs serial amide linkages of amino acid residues by ribosomal complexes. In general, the fidelity of this process is dependent on both accurate recognition of mRNA codons by aminoacyl tRNAs and maintenance of the corresponding open reading frame. However, in a growing number of cases, deviations from this triplet codon rule are observed, indicating that the information content of an mRNA to encode protein may extend beyond its primary structure. These cases, collectively referred to as translational recoding (for review, see Gesteland and Atkins 1996), present exceptions to the venerable genetic code and are typically subdivided among three primary mechanisms: (1) frameshifting, in which translating ribosomes are induced to slide one nucleotide forward or backward at a distinct point in the transcript, with protein synthesis then continuing in the +1 or 11 reading frame, respectively; (2) alternative codon usage, where stop codons are not interpreted as sites of translational termination but, rather, encode an amino acid residue; and (3) translational bypassing, where the translational machinery traverses a gap in the mRNA coding sequence yet yields a single polypeptide chain. Programmed 11 ribosomal frameshift signals are among the most extensively characterized of these translational recoding phenomena (for review, see Brierly 1995; Dinman 1995; Farabaugh 1996). Although current examples are largely limited to viral systems, some bacterial 11 frameshifting events have also been documented (Blinkowa and Walker 1990; Tsuchihashi and Brown 1992; Chandler and Fayet 1993; Engelberg-Kulka and Schoulaker-Schwarz 1994). At present, no examples of eukaryotic mRNAs exhibiting 11 frameshift activity have been reported. However, a number of viruses infecting eukaryotic cells utilize programmed 11 ribosomal frameshifts, demonstrating that the cis elements involved in the frameshifting process are operational in eukaryotes. In viral systems, the efficiency of frameshifting is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus (Brierly 1995 and references therein; Dinman and Wickner 1995). In work presented in this issue, (Hammell et al. 1999), a bioinformatic approach was used to screen prokaryotic and eukaryotic DNA sequence databases for potential 11 frameshift signals. Because of the complexity of 11 frameshift sites and the sequence variability observed among these elements in nature, a multicomponent search algorithm was required to identify candidate sites from bulk database entries. This was made possible in part by the plethora of information available describing structural features of frameshift signals, much of which is described in Hammell et al. (1999) and elsewhere (Brierly 1995; Chen et al. 1996; Marczinke et al. 1998). This strategy represents a marked contrast to many examples of database searching, which typically involve a single motif or are limited to primary structure parameters (Fickett 1996; Altschul et al. 1997; Aravind and Landsman 1998; O’Neill 1998). Database searches using this compound algorithm for 11 frameshift sites yielded a host of potential signals in all databases tested, including eukaryotic sequences, with frequencies significantly higher than found in random sequence populations. In a number of cases, frameshift signals were conserved in homologous mRNAs from different species. A convincing argument for the validity of these searches was provided by the functional demonstration of 11 frameshifting activity for two selected signals (from Saccharomyces cerevisiae RAS1 and human CCR5 mRNAs) in a recombinant assay system. Furthermore, in four cases, potential 11 frameshift signals colocalized to sites of mutation linked to heritable diseases in humans, raising the possibility that 11 frameshift activity may be a physiologically relevant component of regulated gene expression in humans. Modifications of the parameters employed in this search algorithm may yet reveal additional candidate frameshift signals, as the potential for 38-RNA pseudoknot formation was a constraint placed upon their selection in this study. An RNA pseudoknot is not requisite for 11 frameshifting at the gag–pol overlap of HIV-1 (Jacks et al. 1988), for example, which only contains a weak 38-stem–loop structure in vivo (Parkin et al. 1992). The identification of functional 11 frameshift signals in chromosomally encoded eukaryotic mRNAs raises several interesting questions for further investigation. First, it will be important to determine if ribosomal frameshifting occurs in vivo in the context of these transcripts. If so, what is the function of the frameshift event? A ribosomal frameshift may serve to generate alternate protein isoforms, with common aminoCorresponding author. E-MAIL gbrewer@wfubmc.edu; FAX (336) 7169928. Insight/Outlook

Complete Sequence (20 Kilobases) of the Polyprotein-Encoding Gene 1 of Transmissible Gastroenteritis Virus

The entire nucleotide sequence of cloned cDNAs containing the 5′-untranslated region and gene 1 of Purdue-115 strain of transmissible gastroenteritis virus (TGEV) was determined. This completes the sequence of the TGEV genome, which is 28,579 nucleotides long. The gene 1 is composed of two large open reading frames, ORF1a and ORF1b, which contain 4017 and 2698 codons, respectively (stop excluded). A brief, three-codon-long ORF is present upstream of ORF1a. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. In vitro experiments indicated that translation of the ORF1a/b polyprotein involves an efficient ribosomal frameshifting activity, as previously shown for other coronaviruses. Analysis of the predicted ORF1a and ORF1b translation products revealed that the putative functional domains identified in infectious bronchitis virus (IBV), mouse hepatitis virus (MHV) and human coronavirus 229E (HCV 229E) are all present in TGEV. The amino-terminal half of the ORFla product exhibits greater divergence then the carboxy-terminal half, including within the TGEV/HCV229E pair. The ORF1b protein is overall highly conserved among the above four coronaviruses, except a divergent region situated near the carboxy terminus.

Deficiency of 1-Methylguanosine in tRNA from Salmonella typhimurium Induces Frameshifting by Quadruplet Translocation

The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3 -induced frameshift involving mutant tRNAPro and tRNALeu species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid β-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNAPro deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3 -dependent +1 frameshifting. Of the four potential sites for tRNALeu (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNALeu may also act as +1 frameshift suppressors. No - 1 frameshifting activity was found with the trmD3 mutant.

Morphogenic Capabilities of Human Immunodeficiency Virus Type 1 gag and gag-pol Proteins in Insect Cells

To determine the interaction between the gag precursor and the viral protease and to confirm the role of gag precursor in formation of human immunodeficiency virus type 1 particles, the gag and protease encoding regions of a proviral genome with mutations at the site between p17 and p24 or p24 and p15 were expressed by recombinant baculoviruses under the transcriptional control of the strong polyhedrin promoter. Western blot analyses of the expressed products of p17-p24 mutated viruses revealed that both 41- and 55-kDa proteins were synthesized. However, free p24, p17, and the other smaller cleavage products (p9, p6) could not be detected in infected insect cells. The second recombinant virus (p24-p15) synthesized not only a 55k-Da protein, but also a number of smaller products including a 40k-Da protein, p24, and p17. Examination of the insect cells infected by either of these two recombinant viruses by electron microscopy failed to detect any gag particle formation, although some irregular membrane protrusions and profound distortions of the cell surface were clearly visible in the cells infected with recombinant mutant p17-p24 virus, but not with recombinant p24-p15 mutants. To investigate the morphogenic capability of the gag-pol fusion protein, a mutant gag-pol gene containing an inactive protease as well as a modified gag-pol gene lacking the frameshifting activity were expressed in insect cells. While the inactive protease mutant was capable of forming immature particles that were secreted, the frameshifting mutant synthesized only an aberrant form of gag particles with a large radius of curvature in lieu of spherical particles. However, when this mutant was expressed in insect cells in the presence of a truncated gag protein with Mr of 46 kDa (lacking only the p6 domain), normal immature particles containing both antigens were formed.

Acridine structure correlated with mutagenic activity in salmonella

The structural basis for direct mutagenicity of acridines was studied by testing 50 different analogs in the Ames Salmonella tester strains without the addition of mammalian activating enzymes. These experiments showed that the single most effective substituent for frameshift mutagenesis in strain TA1537 is an amino group at the “9” position, while an amino group at either the “3” or “1” position is less effective. Other substitutions at the “9” position demonstrate decreased frameshifting activity compared to 9-aminoacridine. Furthermore, all substituents in combination with the amino group of 9-aminoacridine also decrease frameshifting activity, except for the addition of another amino group at the “1” position or a methyl at the ring nitrogen. Nitro substituents at the “1” and “3” positions enhance 9-aminoacridine toxicity. All nitro substituents decrease typical acridine-frameshift mutagenesis for strain TA1537, but they induce mutagenic activity either in the other type of frameshift strain, TA1538, or in the base-pair substitution strain TA1535. These studies have provided important structure-function relationships for acridine mutagenicity and toxicity in Salmonella. Consequently, this biological system has provided a sensitive means for determining the structural requirements for mutagenic mechanisms.