scholarly journals Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

2001 ◽  
Vol 21 (24) ◽  
pp. 8657-8670 ◽  
Author(s):  
Harry Kontos ◽  
Sawsan Napthine ◽  
Ian Brierley
Keyword(s):  
Rna Structure ◽  
Phase Dependence ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Rna Pseudoknot ◽  
Ribosomal P ◽  
Slippery Sequence ◽  
The Impact ◽  
Ribosomal Pausing

ABSTRACT Here we investigated ribosomal pausing at sites of programmed −1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot.

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1517
Author(s):  
Rebecca S. Brown ◽  
Lisa Kim ◽  
Margaret Kielian
Keyword(s):  
Capsid Protein ◽  
Rna Structure ◽  
Semliki Forest Virus ◽  
Virus Assembly ◽  
Specific Binding ◽  
Genomic Rna ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Labeled Rna

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

scholarly journals Characterization of RNA Elements That Regulate Gag-Pol Ribosomal Frameshifting in Equine Infectious Anemia Virus

2003 ◽  
Vol 77 (19) ◽  
pp. 10280-10287 ◽  
Author(s):  
Chaoping Chen ◽  
Ronald C. Montelaro
Keyword(s):  
Viral Replication ◽  
Rna Structure ◽  
Equine Infectious Anemia Virus ◽  
Stop Codon ◽  
Structural Determinants ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Anemia Virus ◽  
Antiviral Targets ◽  
Slippery Sequence

ABSTRACT Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a −1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed −1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

A Stable Upstream Stem-loop Structure Enhances Selection of the First 5′-ORF-AUG as a Main Start Codon for Translation Initiation of Human ACAT1 mRNA

2004 ◽  
Vol 36 (4) ◽  
pp. 259-268 ◽  
Author(s):  
Li Yang ◽  
Jiang Chen ◽  
Catherine C. Y. Chang ◽  
Xin-Ying Yang ◽  
Zhen-Zhen Wang ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Start Codon ◽  
Loop Structure ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Codon Selection ◽  
Mutation Analyses ◽  
Enzymatic Activity Assay ◽  
Selection Of

Abstract Human ACAT1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG1397–1399 and AUG1415–1417, at 5′-terminus of the open reading frame (ORF, nt 1397–3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5′-terminus of the predicted ORF (5′-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5′-ORF-AUGs can be selected as start codons but the first 5′-ORF-AUG1397–1399 is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5′-ORF-AUG1397–1399 as a main start codon, in addition to upstream nucleotide A in the –3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5′-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5′-ORF-AUGs.

scholarly journals Functional Genetic Elements for Controlling Gene Expression inCupriavidus necatorH16

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Swathi Alagesan ◽  
Erik K. R. Hanko ◽  
Naglis Malys ◽  
Muhammad Ehsaan ◽  
Klaus Winzer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dynamic Range ◽  
Loop Structure ◽  
Stem Loop ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Genetic Elements ◽  
Stem Loop Structure ◽  
Inducible Systems ◽  
The Impact

ABSTRACTA robust and predictable control of gene expression plays an important role in synthetic biology and biotechnology applications. Development and quantitative evaluation of functional genetic elements, such as constitutive and inducible promoters as well as ribosome binding sites (RBSs), are required. In this study, we designed, built, and tested promoters and RBSs for controlling gene expression in the model lithoautotrophCupriavidus necatorH16. A series of variable-strength, insulated, constitutive promoters exhibiting predictable activity within a >700-fold dynamic range was compared to the native PphaC, with the majority of promoters displaying up to a 9-fold higher activity. Positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were evaluated. By supplying different concentrations of inducers, a >1,000-fold range of gene expression levels was achieved. Application of inducible systems for controlling expression of the isoprene synthase geneispSled to isoprene yields that exhibited a significant correlation to the reporter protein synthesis levels. The impact of designed RBSs and other genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was also evaluated. A second-order polynomial relationship was observed between the RBS activities and isoprene yields. This report presents quantitative data on regulatory genetic elements and expands the genetic toolbox ofC. necator.IMPORTANCEThis report provides tools for robust and predictable control of gene expression in the model lithoautotrophC. necatorH16. To address a current need, we designed, built, and tested promoters and RBSs for controlling gene expression inC. necatorH16. To answer a question on how existing and newly developed inducible systems compare, two positively (AraC/ParaBAD-l-arabinose and RhaRS/PrhaBAD-l-rhamnose) and two negatively (AcuR/PacuRI-acrylate and CymR/Pcmt-cumate) regulated inducible systems were quantitatively evaluated and their induction kinetics analyzed. To establish if gene expression can be further improved, the effect of genetic elements, such as mRNA stem-loop structure and A/U-rich sequence, on gene expression was evaluated. Using isoprene production as an example, the study investigated if and to what extent chemical compound yield correlates to the level of gene expression of product-synthesizing enzyme.

scholarly journals The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

2019 ◽  
Vol 48 (2) ◽  
pp. 830-846 ◽  
Author(s):  
Rita Pinto ◽  
Cathrine B Vågbø ◽  
Magnus E Jakobsson ◽  
Yeji Kim ◽  
Marijke P Baltissen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Structure ◽  
Mrna Translation ◽  
28S Rrna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reversible Modification ◽  
And Shifts ◽  
And Function

Abstract RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem–loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

scholarly journals Bacteriophage P4 sut1: a mutation suppressing transcription termination

2007 ◽  
Vol 88 (3) ◽  
pp. 1041-1047
Author(s):  
Susanna Terzano ◽  
Ilaria Oliva ◽  
Francesca Forti ◽  
Claudia Sala ◽  
Francesca Magnoni ◽  
...  
Keyword(s):  
Stop Codon ◽  
Transcription Termination ◽  
Spontaneous Mutation ◽  
Wild Type ◽  
Stem Loop ◽  
Reading Frame ◽  
E Coli ◽  
Stem Loop Structure ◽  
Bacteriophage P4 ◽  
Sense Codon

In the Escherichia coli satellite phage P4, transcription starting from PLE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at PLE and PLL promoters, respectively, and terminating at the intracistronic t151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and α genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 transcription termination site within the 7633–7361 region between orf151 and gene cnr. A potential stem–loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414–7385.

scholarly journals A Group IIC-Type Intron Interrupts the rRNA Methylase Gene of Geobacillus stearothermophilus Strain 10

2010 ◽  
Vol 192 (19) ◽  
pp. 5245-5248 ◽  
Author(s):  
Samuel E. Moretz ◽  
Bert C. Lampson
Keyword(s):  
Open Reading Frame ◽  
Loop Structure ◽  
Geobacillus Stearothermophilus ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Insertion Sites ◽  
Methylase Gene

ABSTRACT Group IIC introns insert next to the stem-loop structure of rho-independent transcription terminators, thus avoiding intact genes. The insertion sites of 17 copies of the G.st.I1 intron from Geobacillus stearothermophilus were compared. One copy of the intron was found to interrupt an open reading frame (ORF) encoding an rRNA methylase.

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