8.6 Reading Frames

The biologist knows that, given a sequence of DNA, it is necessary to examine all six reading frames of the DNA to find the coding regions the cell uses to make proteins.

8.6.1 What Are Reading Frames?

Very often you won't know where in the DNA you're studying the cell actually begins translating the DNA into protein. Only about 1-1.5% of human DNA is in genes, which are the parts of DNA used for the translation into proteins. Furthermore, genes very often occur in pieces that are spliced together during the transcription/translation process.

If you don't know where the translation starts, you have to consider the six possible reading frames. Since the codons are three bases long, the translation happens in three "frames," for instance starting at the first base, or the second, or perhaps the third. (The fourth would be the same as starting from the first.) Each starting place gives a different series of codons, and, as a result, a different series of amino acids.

Also, transcription and translation can happen on either strand of the DNA; that is, either the DNA sequence, or its reverse complement, might contain DNA code that is actually translated. The reverse complement can also be read in any one of three frames. So a total of six reading frames have to be considered when looking for coding regions , that part of the DNA that encodes proteins.

It is therefore quite common to examine all six reading frames of a DNA sequence and to look at the resulting protein translations for long stretches of amino acids that lack stop codons.

The stop codons are definite breaks in the DNAprotein translation process. During translation (actually of RNA to protein, but I'm being deliberately informal and vague about the biochemistry), if a stop codon is reached, the translation stops, and the growing peptide chain grows no more.

Long stretches of DNA that don't contain any stop codons are called open reading frames (ORFs) and are important clues to the presence of a gene in the DNA under study. So gene finder programs need to perform the type of reading frame analysis we'll do in this chapter.

8.6.2 Translating Reading Frames

Based on the facts just presented, let's write some code that translates the DNA in all six reading frames.

In the real world, you'd look around for some subroutines that are already written to do that task. Given the basic nature of the task—something anyone who studies DNA has to do—you'd likely find something. But this is a tutorial, not the real world, so let's soldier on.

This problem doesn't sound too daunting. So, take stock of the subroutines at your disposal, think of where you are and how you can get to your destination.

Looking through the subroutines we've already written, recall dna2peptide. You may recall considering adding some arguments to specify starting and end points. Let's do this now.

Remember that although we calculated reverse complements back in Chapter 4, we never made a subroutine out of it. So let's start there:

# revcom 
#
# A subroutine to compute the reverse complement of DNA sequence

sub revcom {

    my($dna) = @_;

    # First reverse the sequence
    my($revcom) = reverse($dna);

    # Next, complement the sequence, dealing with upper and lower case
    # A->T, T->A, C->G, G->C
    $revcom =~ tr/ACGTacgt/TGCAtgca/;

    return $revcom;
}

Now, a little pseudocode to sketch an idea for the subroutine that will translate specific ranges of DNA:

Given DNA sequence

subroutine translate_frame ( DNA, start, end)

    return dna2peptide( substr( DNA, start, end - start + 1 ) )

}

That went well! Luckily, the substr built-in Perl function made it easy to apply the desired start and end points, while passing the DNA into the already written dna2peptide subroutine.

Note that the length of the sequence is end-start+1. To give a small example: if you start at position 3 and end at position 5, you've got the bases at positions 3, 4, and 5, three bases in all, which is exactly what 5 - 3 + 1 equals.

Dealing with indices like this has to be done carefully, or the code won't work. For many programs, this is the worst the mathematics gets.

Pay attention to the indices!

You have to decide if you wish to keep the numbering of positions from 0, which is Perl's way to do it, or the first character of the sequence is in position 1, which is the biologist's way to do it. Let's do it the biologist's way. The positions will be decreased by one when passed to the Perl function substr, which, of course, does it Perl's way.

The corrected pseudocode looks like this:

Given DNA sequence

subroutine translate_frame ( DNA, start, end)

    # start and end are numbering the sequence from 1 to length

    return dna2peptide( substr( DNA, start - 1, end - start + 1 ) )
}

The length of the desired sequence doesn't change with the change in indices, since:

 (end - 1) - (start - 1) + 1 = end - start + 1

So let's write this subroutine:

# translate_frame
#
# A subroutine to translate a frame of DNA

sub translate_frame {

    my($seq, $start, $end) = @_;

    my $protein;

    # To make the subroutine easier to use, you won't need to specify
    #  the end point--it will just go to the end of the sequence
    #  by default.
    unless($end) {
        $end = length($seq);
    }

    # Finally, calculate and return the translation
        return dna2peptide ( substr ( $seq, $start - 1, $end -$start + 1) );
}

Example 8-4 translates the DNA in all six reading frames.

Example 8-4. Translate a DNA sequence in all six reading frames

#!/usr/bin/perl
# Translate a DNA sequence in all six reading frames

use strict;
use warnings;
use BeginPerlBioinfo;     # see Chapter 6 about this module

# Initialize variables
my @file_data = (  );
my $dna = '';
my $revcom = '';
my $protein = '';

# Read in the contents of the file "sample.dna"
@file_data = get_file_data("sample.dna");

# Extract the sequence data from the contents of the file "sample.dna"
$dna = extract_sequence_from_fasta_data(@file_data);

# Translate the DNA to protein in six reading frames
#   and print the protein in lines 70 characters long
print "\n -------Reading Frame 1--------\n\n";
$protein = translate_frame($dna, 1);
print_sequence($protein, 70);

print "\n -------Reading Frame 2--------\n\n";
$protein = translate_frame($dna, 2);
print_sequence($protein, 70);

print "\n -------Reading Frame 3--------\n\n";
$protein = translate_frame($dna, 3);
print_sequence($protein, 70);

# Calculate reverse complement
$revcom = revcom($dna);

print "\n -------Reading Frame 4--------\n\n";
$protein = translate_frame($revcom, 1);
print_sequence($protein, 70);

print "\n -------Reading Frame 5--------\n\n";
$protein = translate_frame($revcom, 2);
print_sequence($protein, 70);

print "\n -------Reading Frame 6--------\n\n";
$protein = translate_frame($revcom, 3);
print_sequence($protein, 70);

exit;

Here's the output of Example 8-4:

 -------Reading Frame 1--------

RWRR_GVLGALGRPPTGLQRRRRMGPAQ_EYAAWEA_LEAEVVVGAFATAWDAAEWSVQVRGSLAGVVRE
CAGSGDMEGDGSDPEPPDAGEDSKSENGENAPIYCICRKPDINCFMIGCDNCNEWFHGDCIRITEKMAKA
IREWYCRECREKDPKLEIRYRHKKSRERDGNERDSSEPRDEGGGRKRPVPDPDLQRRAGSGTGVGAMLAR
GSASPHKSSPQPLVATPSQHHQQQQQQIKRSARMCGECEACRRTEDCGHCDFCRDMKKFGGPNKIRQKCR
LRQCQLRARESYKYFPSSLSPVTPSESLPRPRRPLPTQQQPQPSQKLGRIREDEGAVASSTVKEPPEATA
TPEPLSDEDL

 -------Reading Frame 2--------

DGGAEGSWGL_AGHLLVCSGDDAWGLRNRSTLPGRRD_KRK_LWAPLQPPGTPPSGLCRFAGRWRGS_GS
APGAEIWREMVQTQSLQMPGRTASPRMGRMRPSTASAANRTSTAS_SGVTTAMSGSMGTASGSLRRWPRP
SGSGTVGSAERKTPS_RFAIGTRSHGSGMAMSGTAVSPGMRVEGARGLSLIQTCSAGQGQGQGLGPCLLG
ALLRPTNPLRSPWWPHPASITSSSSSRSNGQPACVVSVRHVGALRTVVTVISVGT_RSSGAPTRSGRSAG
CASASCGPGNRTSTSLPRSHQ_RPQSPCQGPAGHCPPNSSHSHHRS_GASVKMRGQWRHQQSRSLLRLQP
HLSHSQMRT

 -------Reading Frame 3--------

MAALRGLGGSRPATYWFAAETTHGACAIGVRCLGGVTRSGSSCGRLCNRLGRRRVVCAGSRVAGGGREGV
RRERRYGGRWFRPRASRCRGGQQVREWGECAHLLHLPQTGHQLLHDRV_QLQ_VVPWGLHPDH_EDGQGH
PGVVLSGVQRERPQARDSLSAQEVTGAGWQ_AGQQ_APG_GWRAQEACP_SRPAAPGRVRDRGWGHACSG
LCFAPQILSAALGGHTQPASPAAAAADQTVSPHVW_V_GMSAH_GLWSL_FLSGHEEVRGPQQDPAEVPA
APVPAAGPGIVQVLPFLALTSDALRVPAKAPPATAHPTAATAITEVRAHP_R_GGSGVINSQGAS_GYSH
T_ATLR_GP

 -------Reading Frame 4--------

_VLI_EWLRCGCSLRRLLDC_  _RHCPLIFTDAP_LL_WLWLLLGGQWPAGPWQGL_GRHW_ERGREVLVR
FPGPQLALAQPALLPDLVGAPELLHVPTEITVTTVLSAPTCLTLTTHAG_PFDLLLLLLVMLAGCGHQGL
RRGFVGRSRAPSKHGPNPCP_PCPALQVWIRDRPLAPSTLIPGLTAVPLIAIPLP_LLVPIANL_LGVFL
SALPTVPLPDGLGHLLSDPDAVPMEPLIAVVTPDHEAVDVRFAADAVDGRILPILGLAVLPGIWRLWV_T
ISLHISAPGALPHDPRQRPANLHRPLGGVPGGCKGAHNYFRF_SRLPGSVLLLRRPHASSPLQTSRWPA_
SPQDPSAPPS

 -------Reading Frame 5--------

RSSSESGSGVAVASGGSLTVDDATAPSSSRMRPNFCDGCGCCWVGSGRRGLGRDSEGVTGESEEGKYLYD
SRARSWHWRSRHFCRILLGPPNFFMSRQKSQ_PQSSVRRHASHSPHMRADRLICCCCCW_CWLGVATKGC
GEDLWGEAEPRASMAPTPVPDPARRCRSGSGTGLLRPPPSSRGSLLSRSLPSRSRDFLCR_RISSLGSFS
LHSRQYHSRMALAIFSVIRMQSPWNHSLQLSHPIMKQLMSGLRQMQ_MGAFSPFSDLLSSPASGGSGSEP
SPSISPLPAHSLTTPASDPRTCTDHSAASQAVAKAPTTTSASSHASQAAYSYCAGPMRRLRCKPVGGRPR
APKTPQRRH

 -------Reading Frame 6--------

GPHLRVAQVWL_PQEAP_LLMTPLPPHLHGCALTSVMAVAAVGWAVAGGALAGTLRASLVRARKGSTCTI
PGPAAGTGAAGTSAGSCWGPRTSSCPDRNHSDHSPQCADMPHTHHTCGLTV_SAAAAAGDAGWVWPPRAA
ERICGAKQSPEQAWPQPLSLTLPGAAGLDQGQASCALHPHPGAHCCPAHCHPAPVTSCADSESLAWGLSL
CTPDSTTPGWPWPSSQ_SGCSPHGTTHCSCHTRS_SS_CPVCGRCSRWAHSPHSRTCCPPRHLEALGLNH
LPPYLRSRRTPSRPPPATREPAQTTRRRPRRLQRRPQLLPLLVTPPRQRTPIAQAPCVVSAANQ_VAGLE
PPRPLSAAI