Question

Questions 11-15: Gene structure/Splicing problem. "Protein X" consists of a total of 431 amino acids. Your colleague, techniques) the a biochemist, has purified the protein and determined (via complicated and messy chemical sequence of the first 37 amino acids in the protein, which she has reported to you as follows: HN- MSNITVDDELNLSREQQGFAEDDFIVIKEERETSLSP . nwhile, you have isolated a genomic clone of the gene that codes for protein X, and determined the DNA equence of the first 227 bases from the 5'end of the gene/transcription unit, as shown below (only the coding/non-template strand is indicated). You know that the ORF begins within this sequence (i.e., that it includes the actual start codon), and comparisons with a cDNA clone have indicated that there is a single intron contained within this region (there also others further downstream, but those are not relevant h 60 5'-GTCTTCCACATCGGCCTCACCCATGAGCAACATAACCGTGGATGACGAGCTCAACTTAAG 61 120 CAGAGAACAGCAAGGTGAGTTCAAGTTCAAAACTATATGAATATACTAGCCGTGCTGATT 121 180 GTCTCTTCCTTCCTTTTTCAGGCTTTGCTGAAGACGATTTCATAGTGATCAAGGAGGAGC 181 227 GCGAGACAAGTCTCTCCCCCATAAGGACTACGCACCCGCAGTTTATA.... 3' Questions 11-13: Based on the information presented above (and relevant info from your textbook, including the genetic code and the splice site consensus sequences), you should be able to: 1) relate the amino acid sequence to the DNA sequence, and thereby 2) unambiguously identify the intron vs. exon sequences in the genomic DNA. After doing so, answer the following: 11) How large is the intron (give the exact length/number of bases)? 12) What is the number of the FIRST base in the intron (i.e., the base at/adjacent to the 5' splice site)? (Give the exact number, using the numbering system indicated on the figure above). 13) What is the number of the "branchpoint" nucleotide? (i.e., the invariant "A" which gets linked to the 5' end of the intron in the "lariat" when the RNA is spliced.) Questions 14-15: A transition mutation that affects the FIRST base in the intron (i.e., as indicated in problem 12) prevents splicing. Determining the amino acid sequence that would result if this unspliced mutant mRNA is translated, and then answer the following: 14) How many amino acids (in total) are present in the mutant polypeptide? 15) What is the last (C-terminal) amino acid in the mutant polypeptide?

Answer 1

Answer11: The intron is 67 bases long and its 5' splice site is AG...GTGAGT while the 3' splice site is CAG..G

Answer 12: the first base of intron is located at position 75 of the given seuqence.

Answer 13: The branch point is located at position 118 in the given sequence.

Answer 14; The mutation will change the GTG to ATG. So no splicing will occur and the number of amino acids can be calculated by counting the number of additional codons.