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PROKARYOTIC BIOLOGY MCB 4403 R. H. REEVES Project I: A Prokaryotic Gene - SAMPLE At the bottom of this page is a sequence of DNA that encodes a typical gene found in the genome of Escherichia coli. The nucleotide sequence shown represents the “sense” strand which is complimentary to and in the opposite direction of the template strand. In other words the given sequence of this DNA is the same as the mRNA sequence. The sequence is given in GenBank format; it is presented in lines of 60 nucleotides, separated in groups of ten, and numbered on the left for easy identification. Within this sequence is the gene and all the signals needed for transcription and translation. You are to answer the questions on the next 2 pages (in the spaces provided) and turn in the assignment on Friday, October 12. To locate sequences use the numbering system as indicated on the left. 1 atatggggca tttactacct gtaccctgat aatatctgct attactacct ttccatcccg 61 gtactgatcg tactgcaaag gcacttgaca ccatgctagg agtaccctat aatggactcg -35 S D -10 +1 Start ORF 121 ctcacgagtt cataggaggu tcctcccatg agctatttaa cggcaacagt taaccagctg 181 ccgcaaaaac cacgtgctcg caaagttgcg aaaagcaacg tgcctgcgct ggaagcatgc 241 gtacgcaaac gtggcgtatg tactcgtgta tatactacca ctcctaaaaa accgaactcc 301 cgcctgcgta aagtatgccg tgttcgtctg actaacggtt tcgaagtgac ttcctacatc 361 agtggtgaag gtcacaacct gcaggagcac tccgtgatcc tgatccgtgg cggtcgtgtt 421 tttgacctcc cgggtgttcg ttaccacacc gtacgtggtg cgcttgactg ctccggcgtt 481 aaagaccgta agcaggctcg ttccaagtat ggcgtgaagc gtcctaaggc ttaatggttc 541 tccgttaact acgcaaagta ggatcagtcg atcctacttt tttttacgac gacgtaatag 601 cgtactattt tcgtacccgg tttcagactt ttgagactac atacggtttt ctactacccc 661 atatggggca tttactacct gtaccctgat aatatctgct attactacct ttccatcccg 721 ggactactga ccatgactaa gctaggacta gactaccatc atatactacg ccatatgcag Stop Terminator Answer all the following questions: [5 points each] TRANSLATION: 1. Where is the gene, the ORF? What is its length in nucleotides and how many amino acids are in its protein product? 148-531(or 534), 384(387) nucleotides (bp) 128 amino acids 2. Where is the start codon? What does it code for? Where is the stop codon? Is it amber, ocher or opal? 3. What are the codons for amino acids 4, 5 and 6 and what are these three amino acids? 4. What is the c-terminal amino acid of this protein? 5. Locate the Shine-Delgarno Sequence (RBS). sequence? 6. You have isolated an E. coli strain with a mutation in your protein gene. The sequence of this strain has a base change at position 318, a C to G change. What is the effect of this mutation on your protein? 7. Another mutant is isolated with an A inserted between position 420 and 421. What is the effect on the protein produced by this mutant gene? What is its nucleotide TRANSCRIPTION: 1. Locate the promoter (-35 box to the +1 nucleotide). the sequence below. 2. Where is the terminator and what is its sequence? 3. Is there any evidence for the regulation of this gene. the evidence and why? 4. What is the length (how many nucleotides) of the transcript? 10 points extra Write out What is 1. An amber nonsense mutation of this gene found in another E. coli strain has been located to codon 75. What is the base change for this mutation (as seen in the mRNA)? What is the base-pair change in the DNA? 2. Locate the GenBank entry with the accession number: X76945 What is the source (taxon, genus/species) of this gene? What does the gene code for? Where is the gene (ORF) within the entry (Use the numbering system in the entry.)? What is the tenth amino acid in the protein, the last amino acid and what are their codons? Locate the position and sequence of the Shine-Delgarno sequence for this gene.