Thermodynamic control of −1 programmed ribosomal frameshifting

Abstract mRNA contexts containing a ‘slippery’ sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the −1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how −1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and −1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA−mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

Download Full-text

The energy landscape of −1 ribosomal frameshifting

Science Advances ◽

10.1126/sciadv.aax6969 ◽

2020 ◽

Vol 6 (1) ◽

pp. eaax6969 ◽

Cited By ~ 13

Author(s):

Junhong Choi ◽

Sinéad O’Loughlin ◽

John F. Atkins ◽

Joseph D. Puglisi

Keyword(s):

Single Molecule ◽

Messenger Rna ◽

Information Transfer ◽

Mechanistic Model ◽

High Rate ◽

Coding Region ◽

Ribosomal Frameshifting ◽

Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

Download Full-text

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4557-4560.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4557-4560

Author(s):

O Bakker ◽

J N Philipsen ◽

B C Hennis ◽

G Ab

Keyword(s):

Dimethyl Sulfate ◽

Upstream Region ◽

Nuclear Factor ◽

Base Pairs ◽

Exonuclease Iii ◽

Factor I ◽

Nuclear Factor I ◽

Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

Download Full-text

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2091-2103.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2091-2103

Author(s):

S Türkel ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Physiological Control ◽

Reading Frame ◽

Negative Region ◽

Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

Download Full-text

CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

10.1101/378331 ◽

2018 ◽

Author(s):

Benjamin L. Duchêne ◽

Khadija Cherif ◽

Jean-Paul Iyombe-Engembe ◽

Antoine Guyon ◽

Joel Rousseau ◽

...

Keyword(s):

Hereditary Disease ◽

Reading Frame ◽

Dystrophin Expression ◽

Dmd Gene ◽

Dystrophin Protein ◽

Further Development ◽

Muscle Biopsies ◽

Complete Deletion

AbstractDuchenne Muscular Dystrophy (DMD), a severe hereditary disease, affecting 1 boy out of 3500, mainly results from the deletion of one or more exons leading to a reading frame shift of the DMD gene that abrogates dystrophin protein synthesis. We used the Cas9 of Staphylococcus aureus (SaCas9) to edit the human DMD gene. Pairs of sgRNAs were meticulously chosen to induce a genomic deletion to not only restore the reading frame but also produced a dystrophin protein with normally phased spectrin-like repeats. The formation of a dystrophin protein with spectrin-like repeats normally phased is not usually obtained by skipping or by deletion of complete exons. This can however be obtained in rare instances where the exon/intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion are at similar positions in the spectrin-like repeat. We used pairs of sgRNAs, targeting exons 47 and 58 and a normal reading frame was restored in 67 to 86% of the resulting hybrid exons in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. The restoration of the DMD reading frame and restoration of the dystrophin expression was also obtained in vivo in the heart of the del52hDMD/mđx. Our results provide a proof-of-principle that SaCas9 could be used to edit the human DMD gene and could be considered for the further development of a therapy for DMD.

Download Full-text

Characterization of a Chlamydia psittaciDNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

Infection and Immunity ◽

10.1128/iai.66.3.1167-1173.1998 ◽

1998 ◽

Vol 66 (3) ◽

pp. 1167-1173 ◽

Cited By ~ 31

Author(s):

Li Zhang ◽

Annemarie L. Douglas ◽

Thomas P. Hatch

Keyword(s):

Promoter Region ◽

Chlamydia Psittaci ◽

Host Cells ◽

Upstream Open Reading Frame ◽

Developmental Cycle ◽

Binding Property ◽

Logarithmic Growth Phase ◽

Reading Frame

ABSTRACT The EUO gene (for early upstream open reading frame) ofChlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crpDNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.

Download Full-text

Glucose-regulated protein (GRP94 and GRP78) genes share common regulatory domains and are coordinately regulated by common trans-acting factors

Molecular and Cellular Biology ◽

10.1128/mcb.9.5.2153-2162.1989 ◽

1989 ◽

Vol 9 (5) ◽

pp. 2153-2162

Author(s):

S C Chang ◽

A E Erwin ◽

A S Lee

Keyword(s):

Gene Promoters ◽

Base Pairs ◽

Gene Encoding ◽

Regulatory Domains ◽

Coordinated Expression ◽

Common Domain ◽

Glucose Regulated Protein ◽

Competition Assays

We isolated the promoter of the human gene encoding the 94,000-dalton glucose-regulated protein (GRP94). The 5'-flanking region important for its expression was identified by deletion analysis. Comparison of the promoters of the genes for GRP78 and GRP94 derived from human, rat, and chicken cells revealed a common domain of 28 base pairs within the putative regulatory regions of both genes. This domain has been shown to interact with protein factors in the promoter of the gene for GRP78. Since the genes for GRP94 and GRP78 are transcriptionally regulated with similar kinetics under a variety of stress conditions, we are interested in examining the possible mechanisms for their coordinated expression. Through in vitro and in vivo competition assays, we found that the protein factors which interact with the promoter of the gene for GRP94 also have affinity for the conserved domain of the promoter of the gene for GRP78. These findings suggest that the genes for GRP94 and GRP78 are coordinately regulated through common trans-acting factors which recognize a common regulatory domain of glucose-regulated protein gene promoters.

Download Full-text

Applications of CRISPR/Cas9 for the Treatment of Duchenne Muscular Dystrophy

Journal of Personalized Medicine ◽

10.3390/jpm8040038 ◽

2018 ◽

Vol 8 (4) ◽

pp. 38 ◽

Cited By ~ 15

Author(s):

Kenji Lim ◽

Chantal Yoon ◽

Toshifumi Yokota

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Wide Spectrum ◽

Membrane Integrity ◽

Dystrophin Gene ◽

Muscle Membrane ◽

Reading Frame ◽

Long Term Treatment

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein that leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

Download Full-text

The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1506 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1506-1516 ◽

Cited By ~ 49

Author(s):

Ying Cui ◽

Jonathan D. Dinman ◽

Terri Goss Kinzy ◽

Stuart W. Peltz

Keyword(s):

Site Selection ◽

Start Site ◽

Wild Type ◽

Virus Propagation ◽

Ribosomal Frameshifting ◽

Reading Frame ◽

Translational Accuracy ◽

Its Gene ◽

Killer Virus

ABSTRACT Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene.mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.

Download Full-text

Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0436 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2893-2903 ◽

Cited By ~ 29

Author(s):

Sarah L. Barker ◽

Linda Lee ◽

B. Daniel Pierce ◽

Lymarie Maldonado-Báez ◽

David G. Drubin ◽

...

Keyword(s):

Late Stage ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Type I ◽

Reading Frame ◽

Rich Domain ◽

C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

Download Full-text

OASL Triggered by Novel Goose Astrovirus via ORF2 Restricts Its Replication

Journal of Virology ◽

10.1128/jvi.01767-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Dan Ren ◽

Tuofan Li ◽

Xinyu Zhang ◽

Xiaohui Yao ◽

Wei Gao ◽

...

Keyword(s):

Immune Response ◽

Reading Frame ◽

Enteric Diseases ◽

C Terminus ◽

Orf2 Protein ◽

High Level ◽

Antiviral Targets ◽

Open Reading Frame 2

ABSTRACT Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo. The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses. IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.

Download Full-text