Assignment 1

A major step forward in figuring out the code was the discovery by Nirenberg in 1961 that a cell-free extract made from E. coli cells could translate RNA added to the extract into proteins. The composition of the newly synthesized proteins could be determined by measuring the incorporation of radioactive amino acids into these proteins as they were translated. In his first experiment he made poly U RNA, using the enzyme polynucleotide phosphorylase, and translated it into a peptide of polyphenylalanine using the cell-free extract. This was definitive proof that RNA could code for the synthesis of proteins and gave the first possible assignment of a nucleotide code to the amino acid it specified.


1 .		Because having each nucleotide code for only one amino acid would allow for only four different amino acids to be incorporated into a protein, it was obvious to researchers that there had to be a conversion between multiple bases and each amino acid. Would two nucleotides at a time be sufficient to provide enough codons to code for all 20 amino acids? Why or why not? How many amino acids could be coded for by codons containing only two nucleotides? Will three nucleotides per codon work? Why or why not? Use TranslationLab and then explain your answers below.

2 .		Once it was determined that codons consisted of three-nucleotide sequences, the specificity of each codon could be determined. Use TranslationLab to determine what poly U codes for by performing the following exercise. Click the Start Experiment button on the input screen of TranslationLab. For each of the four bottles of ribonucleotides that appear, click on the arrow to select the uracil (U) nucleotide. Click the Make RNA button to display the poly U sequence of mRNA that you synthesized. Click Add to Notes to create a record of your experiment. To translate this sequence into amino acids, click on the To Translation Mix button. What does poly U code for? Click Add to Notes to add this peptide sequence to the poly U sequence. Repeat the same procedure to make polynucleotides of each of the other three nucleotides. What amino acids do these polynucleotides code for? Refer to a codon chart. Are the amino acids coded for by the polynucleotides you created consistent with what you would expect based on the codon chart?

3 .		If the code is read two bases at a time, what result would you expect for a polydinucleotide such as AUAUAUAU? Try it and see whether your prediction was correct. From your results can you say whether the code is even or odd? Will you get a different result with UAUAUAUA than you did with AUAUAUAU? This result shows that in these crude extracts translation starts at a random location in the RNA sequence. Translate all possible dinucleotides with TranslationLab. Did you get all of the amino acids? If not, which ones are missing? Did you get any amino acids more than once? Which ones? What does this tell you about the code?

4 .		From what you have already discovered, what do you think will happen if you use a polytrinucleotide such as AAC? Enter your hypothesis below.

5 .		Will ACA or CAA give a different result? From these results can you now tell how many bases there are in a codon? If so, how many are there and how do you know this? Comparing this result with the result from polydinucleotide AC, can you now specify a codon for one of the amino acids incorporated by these templates? If so, which codon and which amino acid go together? By elimination, can you assign another codon–amino acid pair? (Hint: Using what you know now, look back at the dinucleotide experiment with AC.) What is it? Try CAC next. Did the results support your codon assignment? Is there evidence here that one of the amino acids must have more than one codon that codes for it? If so, which one?

6 .		What do you think will happen if you translate a tetranucleotide? Try translating the tetranucleotide CAAG. Did you get the result you expected? Can you now assign a codon to any of the other amino acids that appeared in problem 5? (Don't worry about any new amino acids that showed up here, just solve the codons for the amino acids in problem 5.) If so what are they? Test your assignment with AACG. Did this confirm your results? Using the above data and any other experiments that are necessary, assign amino acids to all possible codons that do not include G or U, only various combinations of A and C.

7 .		Now try AU, AAU, and AUU. Did you notice something different this time? What happened, and how would you explain this unusual result? List any new codon assignments that you were able to make from these experiments. Use tetranucleotides to figure out which amino acids go with the codons that can be produced using only A and U. What unusual result did you see with some of the tetranucleotides and what is your explanation for this result?

8 .		Now try GGG, GGA, GGC, and GGU. What amino acid showed up in all four experiments? Are there any codons shared in common by these four reactions? If not, then what must be true to explain your results? Can you propose a codon or codons for the amino acid that showed up in all four experiments? Do the codons that you've just assigned to this amino acid have anything in common? What is it? Use tetranucleotides to prove that your assignment is correct. Comparing these results with the ones above, can you say whether some positions in the codon are less important than others in specifying which amino acid is coded for?