Answers by: Francisco Roque and Nils Weinhold (Some editing by Rasmus Wernersson and Henrik Nielsen)
Step 1: Basic translation
- How is a STOP codon displayed?
- How is a START codon displayed?
>>> (Strict, i.e. "ATG")
))) (Alternative, i.e. "TTG" and "CTG")
- Does a start-codon always code for Methionine (M)?
Yes - but only if it's actually used as a start codon (see http://www.cbs.dtu.dk/services/VirtualRibosome/instructions.php)
- What is the difference between the two types of start codons?
ATG always codes for Methionine. TTG and CTG code for Met is used as a start codon otherwise they code for Leucine. The strict start codon (ATG) is used in >98% of the human transcripts.
Step 2: Genetic codes
- Did the translation succeed?
No, the translation did not succeed very well as there are many stops in the protein sequence.
- Nothing is wrong with the DNA sequence. Can you come up with some good reasons for the result?
The reason is that mitochondria use a different code than the Standard Genetic Code.
- What is the difference in the use of STOP codons?
Yeast mitochondria don't use TGA, only TAA (and TAG, but that does not occur in your sequence).
- What is the difference in the use of START codons?
ATG, TTG, CTG in standard, ATG, ATA in yeast mitochondria (although in your sequence, there are no alternative start codons).
- Are codons coding for completely new amino acids?
Yes, a few codons have changed their meaning: CTN (CTA, CTC, CTG, CTT) now encode Thr instead of Leu. By following the link to the explanation of the genetic codes (linked both in the exercise and on the VirtualRibosome homepage), the following summary of the difference can be found - "Code 3" is the Yeast Mitochondrial code:
Code 3 Standard
AUA Met M Ile I CUU Thr T Leu L CUC Thr T Leu L CUA Thr T Leu L CUG Thr T Leu L UGA Trp W Ter *
CGA absent Arg R CGC absent Arg R
Step 3: Reading frames
- Yeast has introns in some genes, could this be a major problem in this case?
No, in this case it cannot be the problem, since we are dealing with mRNA, which is by definition already processed (the introns have been removed).
- Can an mRNA molecule contain more sequence than the gene in question? (Can it be longer than the CDS coding for the protein).
Yes, it can contain untranslated regions at both ends, including the 5' cap, and the 3' poly-A tail.
- Why is this?
Because mRNA is translated in one direction, its already a copy of one of the strands of DNA.
- What reading frame is most likely the right one?
Reading frame 3, since it has the longest ORF.
- How does the DNA sequence look?
The displayed sequence is complementary to the input.
- In what direction shall it be read?
Bottom right to top left (5' to 3').
- In what direction shall the protein-sequence be read?
Bottom right to top left (N-terminal to C-terminal).
- How many DNA strings are displayed?
- Why is this?
One is the input DNA sequence, the other is the complementary DNA sequence.
Step 4: ORF finder
- Does the result fit to what you found earlier?
Yes, frame 3.
- Would it make any difference to the result if we had only a partial sequence where the last part of the sequence with the STOP codon is missing?
No - by definition an ORF is an open reading frame - simply a reading frame that is not interrupted; it does not have to end with a stop codon.
- What would happen if the first 50 nucleotides (with the START codon) were missing?
One would find a completely different ORF, on -2. If we relaxed the criteria for the ORF finder to not require a start codon, we could still find the correct ORF in frame 3.