Biol 2334 Molecular Biology Translation Worksheet

You will need to use the genetic code table (Fig. 6-50 or in slides), to answer some (many) of these questions. 1. You are working in the lab one day when one of your colleagues comes to you with a dilemma. She
has been trying to isolate a particular mRNA from fruit flies and she has had only limited success. She shows you a partial RNA sequence that is from the middle of the mRNA she is interested in (see below). She needs to know which reading frame is used to generate a protein. You quickly realize that the answer is obvious. Below, write out the amino acids encoded by the open reading frame (dont forget your ends!), and explain how you know which reading frame is the open one. 5 AGUCUAGGCACUGA 3

2. You have just sequenced a short segment of DNA. You wish to analyze this DNA sequence to determine whether it could encode a protein. 5' TCAATGTAACGCGCTACCCCGGAGCTCTGGGCCCAAATTTCATCCACT- 3' A. Find the longest open reading frame (ORF). Underline it on the sequence above. Remember, there are six possibilities, because DNA is double stranded!

B. Is the DNA strand shown above the template; or nontemplate strand?

C. Transcribe this ORF into mRNA, indicating the 5' and 3' ends.

D. Translate this mRNA into amino acids, indicating the amino (N) and carboxy (C) termini

3. Name the three types of RNA important for translation, and the functions of each.

4. Why is a mutation at the third position of a codon often (but not always) of no consequence to the organism?

5. What are the two steps involved in charging of a tRNA? What is the enzyme that catalyzes this reaction? How many different types of this enzyme are found in the human genome?

6. What are the three tRNA binding sites in the ribosome, and what are their functions?

7. Give a brief overview of the main differences between prokaryotic and eukaryotic translation.

8. You are studying a gene in E. coli that encodes an enzyme. The amino acid sequence is shown below: N-Phe-Asn-Trp-Ala-Gln-Met-Trp-C You recover a series of mutants for this gene that show no enzymatic activity. By isolating the mutant enzyme products, you find the following sequences: Mutant 1: Mutant 2: Mutant 3: N-Phe-Asn-Ser-Ala-Gln-Met-Trp-C N-Phe-Asn-Trp-Ala-C N-Phe- Asn-Trp-Ala-Gln-Cys-Gly-Ser-plus another 5 AAs

A) What is the wild type DNA sequence of the gene that encodes this protein? You only need to write out the open reading frame as found on the coding strand. Yes, there is more than one possibility for this sequence. Use TAG for the stop codon. Dont forget your ends!

B) What is the molecular nature of the mutation for each mutant sequence? For each, write out the mutant DNA sequence, underline or box the mutation (where is the mutant DNA different from the wild type DNA?) and state whether the point mutation can be classified a missense, nonsense or frameshift mutation. Each mutant can be explained by a SINGLE nucleotide change. And remember you are examining the DNA sequence here. (4 pts each, 12 pts total). Mutant 1:

Mutant 2:

Mutant 3: