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I would like to find the identity of a protein, together with the organism name and kingdom.

The given DNA sequence is below:

ACTGTTGTTCGGTGATCATCAGTTGTACAACGTCCTAACAACATCACATGCAATGCTTATGATATTCTTC
TTCATCATGCCAGGCACGATGGCAGGACTAGGCAACTTACTAGTGCCATTCCAGATGAGTGTACCGGAGT
TAGTATTCCCAAAGATTAATAACATCGGTATATGATTTTTAGTATGTGGTCTACTTTTGATTACGGGTTC
ATCTTGGATGGAGGAAGGTTCAGGAACGGCCTGAACCGTCTATCCACCACTAGCGCTCACTGCAAGTCAT
AGCGGACTTGCTGTAGATACGTTCATTATCGCATTGCACATGGCCGGTGCAAGCTCCCTTACAGGAAGCA
TCAACCTTATATGTACAATCGCCTATGCCCGCCGTTCACTCATGGCGATGCTGCAGTCATCACTTTATCC
CTGATCCATTACAATCACTGCAGCGTTACTCATAGGAGTTGTGCCTGTGCTAGCAGGTGCTATCACGATG
CTACTCACTGATAGAAGTTGGAGTACCAGCTTCTATGACAGTTCGGCAGGCGGTGATCCTATGTTGTATC
AGCACTTATTCTGGGTGTTTGGGCATCCAGAAGTCTATATCATCATACTTCCAGTATTCGGTATAGTCAG

I've been asked to translate this DNA sequence into the 6 possible frames which are:

5'3' Frame 1
TVVR-SSVVQRPNNITCNAYDILLHHARHDGRTRQLTSAIPDECTGVSIPKD--HRYMIF
SMWSTFDYGFILDGGRFRNGLNRLSTTSAHCKS-RTCCRYVHYRIAHGRCKLPYRKHQPY
MYNRLCPPFTHGDAAVITLSLIHYNHCSVTHRSCACASRCYHDATH--KLEYQLL-QFGR
R-SYVVSALILGVWASRSLYHHTSSIRYSQ
5'3' Frame 2
LLFGDHQLYNVLTTSHAMLMIFFFIMPGTMAGLGNLLVPFQMSVPELVFPKINNIGI-FL
VCGLLLITGSSWMEEGSGTA-TVYPPLALTASHSGLAVDTFIIALHMAGASSLTGSINLI
CTIAYARRSLMAMLQSSLYP-SITITAALLIGVVPVLAGAITMLLTDRSWSTSFYDSSAG
GDPMLYQHLFWVFGHPEVYIIILPVFGIV
5'3' Frame 3
CCSVIISCTTS-QHHMQCL-YSSSSCQARWQD-ATY-CHSR-VYRS-YSQRLITSVYDF-
YVVYF-LRVHLGWRKVQERPEPSIHH-RSLQVIADLL-IRSLSHCTWPVQAPLQEASTLY
VQSPMPAVHSWRCCSHHFIPDPLQSLQRYS-ELCLC-QVLSRCYSLIEVGVPASMTVRQA
VILCCISTYSGCLGIQKSISSYFQYSV-S
3'5' Frame 1
LTIPNTGSMMI-TSGCPNTQNKC-YNIGSPPAELS-KLVLQLLSVSSIVIAPASTGTTPM
SNAAVIVMDQG-SDDCSIAMSERRA-AIVHIRLMLPVRELAPAMCNAIMNVSTASPL-LA
VSASGG-TVQAVPEPSSIQDEPVIKSRPHTKNHIPMLLIFGNTNSGTLIWNGTSKLPSPA
IVPGMMKKNIISIACDVVRTLYN--SPNNS
3'5' Frame 2
-LYRILEV--YRLLDAQTPRISADTT-DHRLPNCHRSWYSNFYQ-VAS--HLLAQAQLL-
VTLQ-L-WIRDKVMTAASP-VNGGHRRLYI-G-CFL-GSLHRPCAMR--TYLQQVRYDLQ
-ALVVDRRFRPFLNLPPSKMNP-SKVDHILKIIYRCY-SLGILTPVHSSGMALVSCLVLP
SCLA--RRIS-ALHVMLLGRCTTDDHRTT
3'5' Frame 3
DYTEYWKYDDIDFWMPKHPE-VLIQHRITACRTVIEAGTPTSISE-HRDSTC-HRHNSYE
-RCSDCNGSGIK--LQHRHE-TAGIGDCTYKVDASCKGACTGHVQCDNERIYSKSAMTCS
ER-WWIDGSGRS-TFLHPR-TRNQK-TTY-KSYTDVINLWEY-LRYTHLEWH--VA-SCH
RAWHDEEEYHKHCM-CC-DVVQLMITEQQ

I ran jobs on Uniprot for all these sequences. Here are the results:

www.uniprot.org/blast/uniprot/B20171003A7434721E10EE6586998A056CCD0537E082E0FZ
www.uniprot.org/blast/uniprot/B2017100383C3DD8CE55183C76102DC5D3A26728BEBF5BAE
www.uniprot.org/blast/uniprot/B20171003A7434721E10EE6586998A056CCD0537E082E5FP
www.uniprot.org/blast/uniprot/B20171003A7434721E10EE6586998A056CCD0537E082F08D
nothing
www.uniprot.org/blast/uniprot/B20171003F725F458AC8690F874DD868E4ED79B88CE32C4B

But how can I decide the identity of the protein and the name of the organism?

In the first one, the organism is Pacific ridley turtle but in the second one it is Diplonema sp. ATCC 50224. So I can only decide at a kingdom level, but not to the organism, right?

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  • 1
    $\begingroup$ Hi snnlaynnkrdsm, I've edited your question to make it sound less "homeworkey", as I suspect that would put people off answering it. $\endgroup$
    – gringer
    Oct 3 '17 at 4:07
  • 1
    $\begingroup$ To be clear, homework questions are fine on this site. But you should be clear when something is a homework assignment. Accordingly, we often won't provide answers to homework questions outright, but lead you in the right direction. $\endgroup$ Oct 3 '17 at 16:53
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Well, let's see. The first step is to simply run a nucleotide blast (blastn) against the nr (non-redundant) database. I did this using the NCBI blast tool. While, as expected, I got many hits, there was only one hit which matched the entire length of the query sequence perfectly:

Diplonema sp. ATCC 50224 cytochrome c oxidase subunit 1 (cox1) mRNA, complete cds; mitochondrial
Sequence ID: JF698657.1
Length: 1612Number of Matches: 1
Range 1: 141 to 770
Alignment statistics for match #1
Score   Expect  Identities  Gaps    Strand
1164 bits(630)  0.0 630/630(100%)   0/630(0%)   Plus/Plus
Query  1    ACTGTTGTTCGGTGATCATCAGTTGTACAACGTCCTAACAACATCACATGCAATGCTTAT  60
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  141  ACTGTTGTTCGGTGATCATCAGTTGTACAACGTCCTAACAACATCACATGCAATGCTTAT  200

Query  61   GATATTCTTCTTCATCATGCCAGGCACGATGGCAGGACTAGGCAACTTACTAGTGCCATT  120
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  201  GATATTCTTCTTCATCATGCCAGGCACGATGGCAGGACTAGGCAACTTACTAGTGCCATT  260

Query  121  CCAGATGAGTGTACCGGAGTTAGTATTCCCAAAGATTAATAACATCGGTATATGATTTTT  180
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  261  CCAGATGAGTGTACCGGAGTTAGTATTCCCAAAGATTAATAACATCGGTATATGATTTTT  320

Query  181  AGTATGTGGTCTACTTTTGATTACGGGTTCATCTTGGATGGAGGAAGGTTCAGGAACGGC  240
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  321  AGTATGTGGTCTACTTTTGATTACGGGTTCATCTTGGATGGAGGAAGGTTCAGGAACGGC  380

Query  241  CTGAACCGTCTATCCACCACTAGCGCTCACTGCAAGTCATAGCGGACTTGCTGTAGATAC  300
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  381  CTGAACCGTCTATCCACCACTAGCGCTCACTGCAAGTCATAGCGGACTTGCTGTAGATAC  440

Query  301  GTTCATTATCGCATTGCACATGGCCGGTGCAAGCTCCCTTACAGGAAGCATCAACCTTAT  360
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  441  GTTCATTATCGCATTGCACATGGCCGGTGCAAGCTCCCTTACAGGAAGCATCAACCTTAT  500

Query  361  ATGTACAATCGCCTATGCCCGCCGTTCACTCATGGCGATGCTGCAGTCATCACTTTATCC  420
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  501  ATGTACAATCGCCTATGCCCGCCGTTCACTCATGGCGATGCTGCAGTCATCACTTTATCC  560

Query  421  CTGATCCATTACAATCACTGCAGCGTTACTCATAGGAGTTGTGCCTGTGCTAGCAGGTGC  480
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  561  CTGATCCATTACAATCACTGCAGCGTTACTCATAGGAGTTGTGCCTGTGCTAGCAGGTGC  620

Query  481  TATCACGATGCTACTCACTGATAGAAGTTGGAGTACCAGCTTCTATGACAGTTCGGCAGG  540
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  621  TATCACGATGCTACTCACTGATAGAAGTTGGAGTACCAGCTTCTATGACAGTTCGGCAGG  680

Query  541  CGGTGATCCTATGTTGTATCAGCACTTATTCTGGGTGTTTGGGCATCCAGAAGTCTATAT  600
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  681  CGGTGATCCTATGTTGTATCAGCACTTATTCTGGGTGTTTGGGCATCCAGAAGTCTATAT  740

Query  601  CATCATACTTCCAGTATTCGGTATAGTCAG  630
            ||||||||||||||||||||||||||||||
Sbjct  741  CATCATACTTCCAGTATTCGGTATAGTCAG  770

So, we now know the sequence is from the gene for mitochondrial Cytochrome C Oxidase subunit 1 from Diplonema sp. ATCC 50224. That the sequence is mitochondrial, is very important since the mitochondrion of this species has a slightly different genetic code. If you go to the NCBI's page for this sequence (JF698657.1), you will see this line:

/transl_table=4

That tells you it is using translation table 4:

4. The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code (transl_table=4)

    AAs  = FFLLSSSSYY**CCWWLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
  Starts = --MM------**-------M------------MMMM---------------M------------
  Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
  Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
  Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

See how all of your translated reading frames have in-frame stop codons? That's because you translated them using the standard genetic code instead of the correct mitochondrial one. As you can see above, in this code, the codon TGA is not a STOP, as in the standard code, and instead codes for tyrosine (W).

So, if you look at the result of the protein blast you ran on UniProt, you will see:

Query1   LLFGDHQLYNVLTTSHAMLMIFFFIMPGTMAGLGNLLVPFQMSVPELVFPKINNIGI-FL   59
         LLFGDHQLYNVLTTSHAMLMIFFFIMPGTMAGLGNLLVPFQMSVPELVFPKINNIGI FL
F5CI61   LLFGDHQLYNVLTTSHAMLMIFFFIMPGTMAGLGNLLVPFQMSVPELVFPKINNIGIWFL   107

Query60  VCGLLLITGSSWMEEGSGTA-TVYPPLALTASHSGLAVDTFIIALHMAGASSLTGSINLI   118
         VCGLLLITGSSWMEEGSGTA TVYPPLALTASHSGLAVDTFIIALHMAGASSLTGSINLI
F5CI61   VCGLLLITGSSWMEEGSGTAWTVYPPLALTASHSGLAVDTFIIALHMAGASSLTGSINLI   167

Query119 CTIAYARRSLMAMLQSSLYP-SITITAALLIGVVPVLAGAITMLLTDRSWSTSFYDSSAG   177
         CTIAYARRSLMAMLQSSLYP SITITAALLIGVVPVLAGAITMLLTDRSWSTSFYDSSAG
F5CI61   CTIAYARRSLMAMLQSSLYPWSITITAALLIGVVPVLAGAITMLLTDRSWSTSFYDSSAG   227

Query178 GDPMLYQHLFWVFGHPEVYIIILPVFGIV                                  206
         GDPMLYQHLFWVFGHPEVYIIILPVFGIV
F5CI61   GDPMLYQHLFWVFGHPEVYIIILPVFGIV                                  256

The only locations where there isn't a 100% identical match is where a W is aligned to a STOP (- in the Uniprot output). So, your actual protein sequence is the 2nd frame of the 5' to 3' direction which, when translated using the correct code, is:

LLFGDHQLYNVLTTSHAMLMIFFFIMPGTMAGLGNLLVPFQMSVPELVFPKINNIGIWFL
VCGLLLITGSSWMEEGSGTAWTVYPPLALTASHSGLAVDTFIIALHMAGASSLTGSINLI
CTIAYARRSLMAMLQSSLYPWSITITAALLIGVVPVLAGAITMLLTDRSWSTSFYDSSAG
GDPMLYQHLFWVFGHPEVYIIILPVFGIV
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Well if it is not a homework assignment, I would suggest to use blastx. Then you don't need to translate your DNA into protein first, but that will be covered by blastx.

If it is a homework assignment though, the teacher probably wants you to select the best blast hit. So which hit gives the highest score, and most matching (identical) amino acids between the alignment.

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