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Open reading frame

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Sample sequence showing three different possible reading frames. Start codons are highlighted in purple, and stop codons are highlighted in red.

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 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[1] 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).[2] That start codon (not necessarily the first) indicates where translation may start. The transcription termination site 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.[3]

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 is a sequence that has a length divisible by three and is bounded by stop codons.[1][4] 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.

Biological significance[edit]

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 a DNA sequence.[5] 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.[5] 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 a typical protein, where the codon usage of that region matches the frequency characteristic for the given organism's coding regions.[5] Therefore, some authors say that an ORF should have a minimal length, e.g. 100 codons[6] or 150 codons.[5] By itself even a long open reading frame is not conclusive evidence for the presence of a gene.[5]

Short ORFs (sORFs)[edit]

Some short ORFs (sORFs), also named Small open reading frames,[7] usually < 100 codons in length,[8] that lack the classical hallmarks of protein-coding genes (both from ncRNAs and mRNAs) can produce functional peptides.[9] 5’-UTR of about 50% of mammal mRNAs are known to contain one or several sORFs,[10] 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.[11] 64–75% of experimentally found translation initiation sites of sORFs are conserved in the genomes of human and mouse and may indicate that these elements have function.[12] 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.[13]

Six-frame translation[edit]

Since DNA is interpreted in groups of three nucleotides (codons), a DNA strand has three distinct reading frames.[14] 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.[14]

Example of a six-frame translation. The nucleotide sequence is shown in the middle with forward translations above and reverse translations below. Two possible open reading frames with the sequences are highlighted.



The ORF Finder (Open Reading Frame Finder)[15] 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 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[16] 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 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[17] is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with 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 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[18]).


ORFik is a R-package in Bioconductor for finding open reading frames and using Next generation sequencing technologies for justification of ORFs.[19] [20]


orfipy is a tool written in Python / Cython to extract ORFs in an extremely and fast and flexible manner.[21] orfipy can work with plain or gzipped FASTA and FASTQ sequences, and provides several options to fine-tune ORF searches; these include specifying 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.[22]

See also[edit]


  1. ^ a b Sieber P, Platzer M, Schuster S (March 2018). "The Definition of Open Reading Frame Revisited". Trends in Genetics. 34 (3): 167–170. doi:10.1016/j.tig.2017.12.009. PMID 29366605.
  2. ^ Brody LC (2021-08-25). "Stop Codon". National Human Genome Research Institute. National Institutes of Health. Retrieved 2021-08-25.
  3. ^ Slonczewski J, Foster JW (2009). Microbiology: An Evolving Science. New York: W.W. Norton & Co. ISBN 978-0-393-97857-5. OCLC 185042615.
  4. ^ Claverie JM (1997). "Computational methods for the identification of genes in vertebrate genomic sequences". Human Molecular Genetics. 6 (10): 1735–44. doi:10.1093/hmg/6.10.1735. PMID 9300666.
  5. ^ a b c d e Deonier R, Tavaré S, Waterman M (2005). Computational Genome Analysis: an introduction. Springer-Verlag. p. 25. ISBN 978-0-387-98785-9.
  6. ^ Claverie JM, Poirot O, Lopez F (1997). "The difficulty of identifying genes in anonymous vertebrate sequences". Computers & Chemistry. 21 (4): 203–14. doi:10.1016/s0097-8485(96)00039-3. PMID 9415985.
  7. ^ Vakirlis, Nikolaos; Vance, Zoe; Duggan, Kate M.; McLysaght, Aoife (2022). "De novo birth of functional microproteins in the human lineage". Cell Reports. 41 (12): 111808. doi:10.1016/j.celrep.2022.111808. PMC 10073203. PMID 36543139. S2CID 254966620.
  8. ^ Kute, Preeti Madhav; Soukarieh, Omar; Tjeldnes, Håkon; Trégouët, David-Alexandre; Valen, Eivind (2022). "Small Open Reading Frames, How to Find Them and Determine Their Function". Frontiers in Genetics. 12: 796060. doi:10.3389/fgene.2021.796060. PMC 8831751. PMID 35154250.
  9. ^ Zanet J, Benrabah E, Li T, Pélissier-Monier A, Chanut-Delalande H, Ronsin B, et al. (September 2015). "Pri sORF peptides induce selective proteasome-mediated protein processing". Science. 349 (6254): 1356–1358. Bibcode:2015Sci...349.1356Z. doi:10.1126/science.aac5677. PMID 26383956. S2CID 206639549.
  10. ^ Wethmar K, Barbosa-Silva A, Andrade-Navarro MA, Leutz A (January 2014). "uORFdb--a comprehensive literature database on eukaryotic uORF biology". Nucleic Acids Research. 42 (Database issue): D60–D67. doi:10.1093/nar/gkt952. PMC 3964959. PMID 24163100.
  11. ^ Geballe, A. P.; Morris, D. R. (April 1994). "Initiation codons within 5'-leaders of mRNAs as regulators of translation". Trends in Biochemical Sciences. 19 (4): 159–164. doi:10.1016/0968-0004(94)90277-1. ISSN 0968-0004. PMID 8016865.
  12. ^ Lee S, Liu B, Lee S, Huang SX, Shen B, Qian SB (September 2012). "Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution". Proceedings of the National Academy of Sciences of the United States of America. 109 (37): E2424–E2432. doi:10.1073/pnas.1207846109. PMC 3443142. PMID 22927429.
  13. ^ Schwartz AM, Putlyaeva LV, Covich M, Klepikova AV, Akulich KA, Vorontsov IE, et al. (October 2016). "Early B-cell factor 1 (EBF1) is critical for transcriptional control of SLAMF1 gene in human B cells". Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1859 (10): 1259–1268. doi:10.1016/j.bbagrm.2016.07.004. PMID 27424222.
  14. ^ a b Pearson WR, Wood T, Zhang Z, Miller W (November 1997). "Comparison of DNA sequences with protein sequences". Genomics. 46 (1): 24–36. doi:10.1006/geno.1997.4995. PMID 9403055. S2CID 6413018.
  15. ^ "ORFfinder". National Center for Biotechnology Information.
  16. ^ Dhar DV, Kumar MS (2012). "ORF Investigator: A New ORF finding tool combining Pairwise Global Gene Alignment". Research Journal of Recent Sciences. 1 (11): 32–35.
  17. ^ "OrfPredictor". bioinformatics.ysu.edu. Archived from the original on 2015-12-22. Retrieved 2015-12-17.
  18. ^ Carrington JC, Freed DD (April 1990). "Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region". Journal of Virology. 64 (4): 1590–7. doi:10.1128/JVI.64.4.1590-1597.1990. PMC 249294. PMID 2319646.
  19. ^ Kornel Labun, Haakon Tjeldnes (2018). "ORFik - Open reading frames in genomics". bioconductor.org. doi:10.18129/B9.bioc.ORFik.
  20. ^ Tjeldnes, Håkon; Labun, Kornel; Torres Cleuren, Yamila; Chyżyńska, Katarzyna; Świrski, Michał; Valen, Eivind (2021). "ORFik: A comprehensive R toolkit for the analysis of translation". BMC Bioinformatics. 22 (1): 336. doi:10.1186/s12859-021-04254-w. PMC 8214792. PMID 34147079.
  21. ^ Singh U, Wurtele ES (February 2021). "orfipy: a fast and flexible tool for extracting ORFs". Bioinformatics. 37 (18): 3019–3020. doi:10.1093/bioinformatics/btab090. ISSN 1367-4803. PMC 8479652. PMID 33576786.
  22. ^ Singh U (2021-02-13), urmi-21/orfipy, retrieved 2021-02-13

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