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48-500: ORF1a is the first open reading frame at the 5' end of the genome. Together ORF1ab occupies about two thirds of the genome, with the remaining third at the 3' end encoding the structural proteins and accessory proteins . It is translated from a 5' capped RNA by cap-dependent translation . Nidoviruses have a complex system of discontinuous subgenomic RNA production to enable expression of genes in their relatively large RNA genomes (typically 27-32 kb for coronaviruses), but ORF1ab

96-410: A DNA sequence. The presence of an ORF does not necessarily mean that the region is always translated . For example, in a randomly generated DNA sequence with an equal percentage of each nucleotide , a stop-codon would be expected once every 21 codons . A simple gene prediction algorithm for prokaryotes might look for a start codon followed by an open reading frame that is long enough to encode

144-459: A pseudoknot RNA secondary structure . This has been measured at between 20-50% efficiency for murine coronavirus , or 45-70% in SARS-CoV-2 yielding a stoichiometry of roughly 1.5 to 2 times as much pp1a as pp1ab protein expressed. The polyproteins pp1a and pp1ab contain about 13 to 17 nonstructural proteins . They undergo auto- proteolysis to release the nonstructural proteins due to

192-423: A stem-loop or pseudoknot ) is thought to pause the ribosome on the slippery site during translation, forcing it to relocate and continue replication from the −1 position. It is believed that this occurs because the structure physically blocks movement of the ribosome by becoming stuck in the ribosome mRNA tunnel. This model is supported by the fact that strength of the pseudoknot has been positively correlated with

240-444: A +1 frameshift signal does not have the same motif, and instead appears to function by pausing the ribosome at a sequence encoding a rare amino acid. Ribosomes do not translate proteins at a steady rate, regardless of the sequence. Certain codons take longer to translate, because there are not equal amounts of tRNA of that particular codon in the cytosol . Due to this lag, there exist in small sections of codons sequences that control

288-402: A DNA strand has three distinct reading frames. The double helix of a DNA molecule has two anti-parallel strands; with the two strands having three reading frames each, there are six possible frame translations. The ORF Finder (Open Reading Frame Finder) is a graphical analysis tool which finds all open reading frames of a selectable minimum size in a user's sequence or in a sequence already in

336-399: A completely different frame thereafter. In programmed −1 ribosomal frameshifting, the slippery sequence fits a X_XXY_YYH motif, where XXX is any three identical nucleotides (though some exceptions occur), YYY typically represents UUU or AAA, and H is A, C or U. In the case of +1 frameshifting, the slippery sequence contains codons for which the corresponding tRNA is more rare, and the frameshift

384-526: A hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments, otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences. The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where

432-535: A particular nucleotide at a position, there are also letters that represent ambiguity which are used when more than one kind of nucleotide could occur at that position. The rules of the International Union of Pure and Applied Chemistry ( IUPAC ) are as follows: These symbols are also valid for RNA, except with U (uracil) replacing T (thymine). Small molecules, proteins, and nucleic acids have been found to stimulate levels of frameshifting. For example,

480-416: A proper ratio of 0-frame (normal translation) and "trans-frame" (encoded by frameshifted sequence) proteins. Its use in viruses is primarily for compacting more genetic information into a shorter amount of genetic material. In eukaryotes it appears to play a role in regulating gene expression levels by generating premature stops and producing nonfunctional transcripts. The most common type of frameshifting

528-476: A region of mRNA base pairing with another region on the same strand, are shown protruding from the linear DNA. The linear region of the HIV ribosomal frameshift signal contains a highly conserved UUU UUU A slippery sequence; many of the other predicted structures contain candidates for slippery sequences as well. The mRNA sequences in the images can be read according to a set of guidelines. While A, T, C, and G represent

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576-420: A single large ORF encoding a polyprotein of over 13,000 amino acids . In these non-canonical genomes, other frameshift locations or stop codon readthrough may be used to regulate the stoichiometry of viral proteins. Nidoviruses vary widely in genome size, from arteriviruses with typically 12-15kb genomes to coronaviruses at 27-32kb. Their evolutionary history has been of research interest in understanding

624-415: A slippery sequence, an RNA secondary structure, or both. A −1 frameshift signal consists of both elements separated by a spacer region typically 5–9 nucleotides long. Frameshifting may also be induced by other molecules which interact with the ribosome or the mRNA (trans-acting). Slippery sequences can potentially make the reading ribosome "slip" and skip a number of nucleotides (usually only 1) and read

672-493: A tandem slippage model, in which the ribosomal P-site tRNA anticodon re-pairs from XXY to XXX and the A-site anticodon re-pairs from YYH to YYY simultaneously. These new pairings are identical to the 0-frame pairings except at their third positions. This difference does not significantly disfavor anticodon binding because the third nucleotide in a codon, known as the wobble position , has weaker tRNA anticodon binding specificity than

720-452: A typical protein, where the codon usage of that region matches the frequency characteristic for the given organism's coding regions. Therefore, some authors say that an ORF should have a minimal length, e.g. 100 codons or 150 codons. By itself even a long open reading frame is not conclusive evidence for the presence of a gene . Some short ORFs (sORFs), also named Small open reading frames , usually < 100 codons in length, that lack

768-407: A −1 frameshift signal: a slippery sequence , a spacer region, and an RNA secondary structure. The slippery sequence fits a X_XXY_YYH motif, where XXX is any three identical nucleotides (though some exceptions occur), YYY typically represents UUU or AAA, and H is A, C or U. Because the structure of this motif contains 2 adjacent 3-nucleotide repeats it is believed that −1 frameshifting is described by

816-446: Is −1 frameshifting or programmed −1 ribosomal frameshifting (−1 PRF) . Other, rarer types of frameshifting include +1 and −2 frameshifting. −1 and +1 frameshifting are believed to be controlled by different mechanisms, which are discussed below. Both mechanisms are kinetically driven . In −1 frameshifting, the ribosome slips back one nucleotide and continues translation in the −1 frame. There are typically three elements that comprise

864-514: Is a R-package in Bioconductor for finding open reading frames and using Next generation sequencing technologies for justification of ORFs. orfipy is a tool written in Python / Cython to extract ORFs in an extremely and fast and flexible manner. orfipy can work with plain or gzipped FASTA and FASTQ sequences, and provides several options to fine-tune ORF searches; these include specifying

912-581: Is a biological phenomenon that occurs during translation that results in the production of multiple, unique proteins from a single mRNA . The process can be programmed by the nucleotide sequence of the mRNA and is sometimes affected by the secondary, 3-dimensional mRNA structure . It has been described mainly in viruses (especially retroviruses ), retrotransposons and bacterial insertion elements, and also in some cellular genes . Small molecules, proteins, and nucleic acids have also been found to stimulate levels of frameshifting. In December 2023, it

960-489: Is a program which not only gives information about the coding and non coding sequences but also can perform pairwise global alignment of different gene/DNA regions sequences. The tool efficiently finds the ORFs for corresponding amino acid sequences and converts them into their single letter amino acid code, and provides their locations in the sequence. The pairwise global alignment between the sequences makes it convenient to detect

1008-585: Is a sequence that has a length divisible by three and is bounded by stop codons. This more general definition can be useful in the context of transcriptomics and metagenomics , where a start or stop codon may not be present in the obtained sequences. Such an ORF corresponds to parts of a gene rather than the complete gene. One common use of open reading frames (ORFs) is as one piece of evidence to assist in gene prediction . Long ORFs are often used, along with other evidence, to initially identify candidate protein-coding regions or functional RNA -coding regions in

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1056-407: Is favored because the codon in the new frame has a more common associated tRNA. One example of a slippery sequence is the polyA on mRNA, which is known to induce ribosome slippage even in the absence of any other elements. Efficient ribosomal frameshifting generally requires the presence of an RNA secondary structure to enhance the effects of the slippery sequence. The RNA structure (which can be

1104-656: Is located after the ORF, beyond the translation stop codon. If transcription were to cease before the stop codon, an incomplete protein would be made during translation. In eukaryotic genes with multiple exons , introns are removed and exons are then joined together after transcription to yield the final mRNA for protein translation. In the context of gene finding , the start-stop definition of an ORF therefore only applies to spliced mRNAs , not genomic DNA, since introns may contain stop codons and/or cause shifts between reading frames. An alternative definition says that an ORF

1152-407: Is translated directly from the genomic RNA. ORF1ab sequences have been observed in noncanonical subgenomic RNAs, though their functional significance is unclear. A programmed ribosomal frameshift allows reading through the stop codon that terminates ORF1a to continue in a -1 reading frame , producing the longer polyprotein pp1ab. The frameshift occurs at a slippery sequence which is followed by

1200-458: Is translated into a single amino acid . The code itself is considered degenerate , meaning that a particular amino acid can be specified by more than one codon. However, a shift of any number of nucleotides that is not divisible by 3 in the reading frame will cause subsequent codons to be read differently. This effectively changes the ribosomal reading frame . In this example, the following sentence of three-letter words makes sense when read from

1248-512: The COVID-19 pandemic , the genome of SARS-CoV-2 viruses has been sequenced many times, resulting in identification of thousands of distinct variants . In a World Health Organization analysis from July 2020, ORF1ab was the most frequently mutated gene, followed by the S gene encoding the spike protein . The most commonly mutated protein within ORF1ab was papain-like protease (nsp3), and

1296-461: The main protease flanked on either end by transmembrane domains ; and from ORF1b, a nucleotidyltransferase domain known as NiRAN , RNA-dependent RNA polymerase (RdRp), a zinc -binding domain, and a helicase . (This is sometimes considered seven domains, counting the transmembrane regions separately.) In addition, an endoribonuclease domain is found in all nidoviruses that infect vertebrate hosts. Arteriviruses, which have smaller genomes than

1344-630: The six possible reading frames will be "open" (the "reading", however, refers to the RNA produced by transcription of the DNA and its subsequent interaction with the ribosome in translation ). Such an ORF may contain a start codon (usually AUG in terms of RNA ) and by definition cannot extend beyond a stop codon (usually UAA, UAG or UGA in RNA). That start codon (not necessarily the first) indicates where translation may start. The transcription termination site

1392-426: The actions of internal cysteine protease domains . In coronaviruses, there are a total of 16 nonstructural proteins; pp1a protein contains nonstructural proteins nsp1-11 and the pp1ab protein contains nsp1-10 and nsp12-16. Proteolytic processing is performed by two proteases: the papain-like protease protein domain located in the multidomain protein nsp3 cleaves up to nsp4, and the 3CL protease (also known as

1440-437: The associated gene. If a novel or off-target protein is produced, it can trigger other unknown consequences. In viruses this phenomenon may be programmed to occur at particular sites and allows the virus to encode multiple types of proteins from the same mRNA. Notable examples include HIV-1 (human immunodeficiency virus), RSV ( Rous sarcoma virus ) and the influenza virus (flu), which all rely on frameshifting to create

1488-471: The beginning, these codons make sense to a ribosome and can be translated into amino acids (AA) under the vertebrate mitochondrial code : However, let's change the reading frame by starting one nucleotide downstream (effectively a "+1 frameshift" when considering the 0 position to be the initial position of A ): Because of this +1 frameshifting, the DNA sequence is read differently. The different codon reading frame therefore yields different amino acids. In

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1536-430: The beginning: However, if the reading frame is shifted by one letter to between the T and H of the first word (effectively a +1 frameshift when considering the 0 position to be the initial position of T ), then the sentence reads differently, making no sense. In this example, the following sequence is a region of the human mitochondrial genome with the two overlapping genes MT-ATP8 and MT-ATP6 . When read from

1584-420: The case of a translating ribosome, a frameshift can either result in nonsense mutation , a premature stop codon after the frameshift, or the creation of a completely new protein after the frameshift. In the case where a frameshift results in nonsense, the nonsense-mediated mRNA decay (NMD) pathway may destroy the mRNA transcript, so frameshifting would serve as a method of regulating the expression level of

1632-526: The classical hallmarks of protein-coding genes (both from ncRNAs and mRNAs) can produce functional peptides. 5’-UTR of about 50% of mammal mRNAs are known to contain one or several sORFs, also called upstream ORFs or uORFs . However, less than 10% of the vertebrate mRNAs surveyed in an older study contained AUG codons in front of the major ORF. Interestingly, uORFs were found in two thirds of proto-oncogenes and related proteins. 64–75% of experimentally found translation initiation sites of sORFs are conserved in

1680-447: The coding region begins and ends. OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects. ORF Predictor uses a combination of the two different ORF definitions mentioned above. It searches stretches starting with a start codon and ending at a stop codon. As an additional criterion, it searches for a stop codon in the 5' untranslated region (UTR or NTR, nontranslated region ). ORFik

1728-524: The database. This tool identifies all open reading frames using the standard or alternative genetic codes. The deduced amino acid sequence can be saved in various formats and searched against the sequence database using the basic local alignment search tool (BLAST) server. The ORF Finder should be helpful in preparing complete and accurate sequence submissions. It is also packaged with the Sequin sequence submission software (sequence analyser). ORF Investigator

1776-435: The different mutations, including single nucleotide polymorphism . Needleman–Wunsch algorithms are used for the gene alignment. The ORF Investigator is written in the portable Perl programming language , and is therefore available to users of all common operating systems. OrfPredictor is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with

1824-420: The first and second nucleotides. In this model, the motif structure is explained by the fact that the first and second positions of the anticodons must be able to pair perfectly in both the 0 and −1 frames. Therefore, nucleotides 2 and 1 must be identical, and nucleotides 3 and 2 must also be identical, leading to a required sequence of 3 identical nucleotides for each tRNA that slips. The slippery sequence for

1872-495: The following functions: The structure and organization of the genome, including ORF1a, ORF1b, and the frameshift separating them, is conserved among nidoviruses. Some "non-canonical" nidovirus structures have been described, mainly involving gene fusions . The largest known nidovirus, planarian secretory cell nidovirus (PSCNV), with a 41kb genome, has a non-canonical genome structure in which ORF1a, ORF1b, and downstream ORFs containing structural proteins are fused and expressed as

1920-413: The genomes of human and mouse and may indicate that these elements have function. However, sORFs can often be found only in the minor forms of mRNAs and avoid selection; the high conservation of initiation sites may be connected with their location inside promoters of the relevant genes. This is characteristic of SLAMF1 gene, for example. Since DNA is interpreted in groups of three nucleotides (codons),

1968-524: The level of frameshifting for associated mRNA. Below are examples of predicted secondary structures for frameshift elements shown to stimulate frameshifting in a variety of organisms. The majority of the structures shown are stem-loops, with the exception of the ALIL (apical loop-internal loop) pseudoknot structure. In these images, the larger and incomplete circles of mRNA represent linear regions. The secondary "stem-loop" structures, where "stems" are formed by

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2016-767: The main protease, nsp5) performs the remaining cleavages of nsp5 through the polyprotein C-terminus . Proteins nsp12-16, the C-terminal components of the pp1ab polyprotein, contain the core enzymatic activities necessary for viral replication . After proteolytic processing, several of the nonstructural proteins assemble into a large protein complex known as the replicase-transcriptase complex (RTC) which performs genome replication and transcription . A set of five conserved "core replicase" protein domains are present in all nidovirus lineages ( arteriviruses , mesoniviruses , roniviruses , and coronaviruses ): from ORF1a,

2064-430: The other nidovirus lineages, also lack methyltransferases as well as a proofreading exoribonuclease , a domain that is conserved in nidoviruses with larger genomes. This proofreading functionality is thought to be required for sufficient fidelity to replicate large RNA genomes, but may also play additional roles in some viruses. In coronaviruses, pp1a and pp1ab together contain sixteen nonstructural proteins, which have

2112-445: The rate of ribosomal frameshifting. Specifically, the ribosome must pause to wait for the arrival of a rare tRNA, and this increases the kinetic favorability of the ribosome and its associated tRNA slipping into the new frame. In this model, the change in reading frame is caused by a single tRNA slip rather than two. Ribosomal frameshifting may be controlled by mechanisms found in the mRNA sequence (cis-acting). This generally refers to

2160-433: The replication of very large RNA genomes despite the relatively low-fidelity replication mechanism of the viral RNA-dependent RNA polymerase (RdRp). The larger nidovirus genomes (above around 20kb) encode a proofreading exoribonuclease ( nsp14 in coronaviruses) thought to be required for replication fidelity. Among coronaviruses , ORF1ab is more highly conserved than the 3' ORFs encoding structural proteins . Throughout

2208-504: The single most commonly observed missense mutation was in RNA-dependent RNA polymerase . Some PCR tests that detect COVID-19 analyze the specimen for the ORF1ab gene, among others. Open reading frame In molecular biology , reading frames are defined as spans of DNA sequence between the start and stop codons . Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of

2256-452: The start and stop codons, reporting partial ORFs, and using custom translation tables. The results can be saved in multiple formats, including the space-efficient BED format. orfipy is particularly faster for data containing multiple smaller FASTA sequences, such as de-novo transcriptome assemblies. Programmed ribosomal frameshift Ribosomal frameshifting , also known as translational frameshifting or translational recoding ,

2304-413: Was reported that in vitro -transcribed (IVT) mRNAs in response to BNT162b2 (Pfizer–BioNTech) anti-COVID-19 vaccine caused ribosomal frameshifting. Proteins are translated by reading tri-nucleotides on the mRNA strand, also known as codons , from one end of the mRNA to the other (from the 5' to the 3' end ) starting with the amino acid methionine as the start (initiation) codon AUG. Each codon

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