
In
molecular biology
Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
, 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
Translation is the communication of the semantics, meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The English la ...
). Such an open reading frame (ORF) may
contain a
start codon (usually AUG in terms of
RNA
Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
) 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
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
would be made during translation.
In
eukaryotic
The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...
gene
In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
s with multiple
exon
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequence ...
s,
intron
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e., a region inside a gene."The notion of the cistron .e., gen ...
s are removed and exons are then joined together after transcription to yield the final
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
for protein translation. In the context of
gene finding, the start-stop
definition
A definition is a statement of the meaning of a term (a word, phrase, or other set of symbols). Definitions can be classified into two large categories: intensional definitions (which try to give the sense of a term), and extensional definitio ...
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
Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...
, a
stop-codon would be expected once every 21
codons.
A simple gene prediction algorithm for
prokaryotes
A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...
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
In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
.
Short open reading frames
Some short open reading frames, also named small open reading frames, abbreviated as sORFs or smORFs, usually < 100 codons in length, that lack the classical hallmarks of protein-coding genes (both from ncRNAs and mRNAs) can produce functional
peptide
Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...
s.
They encode
microproteins or sORF‐encoded proteins (SEPs). The 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).
Investigator
ORF Investigator 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
Perl is a high-level, general-purpose, interpreted, dynamic programming language. Though Perl is not officially an acronym, there are various backronyms in use, including "Practical Extraction and Reporting Language".
Perl was developed ...
programming language
A programming language is a system of notation for writing computer programs.
Programming languages are described in terms of their Syntax (programming languages), syntax (form) and semantics (computer science), semantics (meaning), usually def ...
, and is therefore available to users of all common operating systems.
Predictor
OrfPredictor 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''
). The OrfPredictor web server was not further supported, the standalone OrfPredictor tool can be downloaded at the following site (http://bioinformatics.ysu.edu/publication/tools_download/).
ORFik
ORFik is a R-package in Bioconductor for finding open reading frames and using Next generation sequencing technologies for justification of ORFs.
orfipy
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 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.
See also
*
Coding region
*
Putative gene
*
Sequerome – A
sequence profiling tool that links each
BLAST record to the
NCBI ORF enabling complete ORF analysis of a BLAST report.
*
Micropeptide
References
External links
Translation and Open Reading FrameshORFeome V5.1- A web-based interactive tool for CCSB Human ORFeome Collection
- A free, fast and multi-platform desktop GUI tool for predicting and analyzing ORFs
StarORF- A multi-platform, java-based, GUI tool for predicting and analyzing ORFs and obtaining reverse complement sequence
{{Webarchive, url=https://web.archive.org/web/20151222082631/http://bioinformatics.ysu.edu/tools/OrfPredictor.html , date=2015-12-22 - A webserver designed for ORF prediction and translation of a batch of EST or cDNA sequences
Molecular genetics
Bioinformatics
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