More Practice Decoding DNA!

The following sequence is the nontemplate strand of the start of the human RPE65 gene found on chromosome 1. To make it easier to read, spaces have been introduced every ten nucleotides. Transcription begins at the first nucleotide shown and RNA Polymerase II moves to the right transcribing this full sequence and beyond. The location of Intron #1 is shown.

actctctgaa ctggaagaaa tgtctatgca ggAtgaêgcat cctTAAggtg gttacêaagaa actgtttgaa actgtggagg TAT

1 11 21 31 41 51 61 71 81

Intron #1

1. Is the left end of this DNA molecule a 5’ or a 3’ end? How do you know?

It’s a 5’ end. Since it’s a nontemplate strand, it will be complementary to the template strand. The mRNA is also complementary to the template strand and thus the sequence of the mRNA will be just like the above sequence (except for T vs. U). If RNA polymerase II is moving left-to-right, the right end must be a 3’ end, since polymerases only ever add to a 3’ end.

2. How did RNA Polymerase II get to this place on chromosome 1 to begin transcribing?

It needed a promoter sequence (consisting of a TATA box).

3. How will the 5’ end the the resulting mRNA be altered?

The 5’ end will be capped.

4. There are four differnet AUG codons in the mRNA. How is the correct start codon selected?

The small subunit of the ribosome binds to the 5’ cap and slides in a 3’ direction until it encounters the first start codon.

5. What are the 15 amino acids at the N-terminus of RPE65?

Met-Ser-Met-Gln-Asp-Glu-Arg-Asn-Cys-Leu-Lys-Leu-Trp-Arg-Tyr…

6. List the anticodons present on the first three tRNAs used to translate RPE65.

5’ CAU 5’ UGU 5’ CAU

7. To help understand this gene and the disease LCA2, we can determine the sequence of many RPE65 alleles from many different people. Allele #1 is the sequence shown above. Allele #2 is just like #1 except that nucleotide 31 is an A instead of the G. What are the first 15 amino acids of the RPE65 protein encoded by this allele? What type of mutation is this?

No change: Met-Ser-Met-Gln-Asp-Glu-Arg-Asn-Cys-Leu-Lys-Leu-Trp-Arg-Tyr…

Silent mutation

8. Allele #3 is just like allele #1 except that nucloetide 60 is a T instead of a G. What are the first 15 amino acids of the RPE65 protein encoded by this allele? What type of mutation is this?

Met-Ser-Met-Gln-Asp-Glu-Arg-Asp-Cys-Leu-Lys-Leu-Trp-Arg-Tyr…

Missense

9. Allele #4 is just like allele #1 except that nucleotides 29 and 30 (CA) have been deleted. What are the first 15 amino acids of the RPE65 protein encoded by this allele? What type of mutation is this?

Met-Ser-Met-Gly-C

Nonsense

10. Allele #5 is just like allele #1 except that one extra T nucleotide has been inserted between nucleotides 41 and 42. What are the first 15 amino acids of the RPE65 protein encoded by this allele?

Met-Ser-Met-Gln-Asp-Glu-Arg-Asn-Cys-Leu-Lys-Leu-Trp-Arg-Tyr…

No change since it’s in an intron

11. Allele #6 is just like allele #1 except that the G at nucleotide 37 is replaced with a C. Due to this change, the intron is not spliced out. What are the first 15 amino acids of the RPE65 protein encoded by this allele?

Met-Ser-Met-Gln-Asp-Glu-His-Pro-C

12. A woman carries allele #1 and allele #4 on her two copies of chromosome 1. Her husband has allele #5 and allele #6. What is the probability that their child will have LCA2?

25%. Each parent is heterozygous and one of each parents’ alleles is nonfunctional. If the kid gets allele #4 from mom and allele #6 from dad, they will have no functional RPE65 enzyme and thus have LCA2. If they get an allele #1 from mom or an allele #5 from dad (or both), they have a functional RPE65 and won’t have the disease.