In molecular biology, open reading frames (ORFs) 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 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 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 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.
Biological significance
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. 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 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.
Short ORFs (sORFs) Some short ORFs (sORFs), also named Small open reading frames, usually < 100 codons in length, that lack 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 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.
Six-frame translation
Since DNA is interpreted in groups of three nucleotides (codons), 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.
Software
Finder
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 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).