Lab 6 - Cracking the Genetic Code - Fall 2023

Laboratory 6: Cracking the Genetic Code 1 LABORATORY 6: CRACKING THE GENETIC CODE Objectives In this laboratory session you will examine some of the experiments used by scientists in the 1960s to crack the genetic code. The procedure involved synthesizing specific strands of messenger RNA (mRNA) and then examining what amino acids were produced by translation of these genetic instructions. This was a complex, puzzle-solving exercise that took many years and deservedly won the Nobel Prize. You will be using a computer simulation to reproduce these experiments and use the same systematic approach to construct your own version of the genetic code. Preparation Carefully read the introduction in order to prepare for this laboratory project and quiz. You should be familiar with the following concepts and techniques:  transcription  translation  codons  amino acid structure  using cell extracts

Laboratory 6: Cracking the Genetic Code 2 INTRODUCTION The Genetic Code Our proteins are polymers of amino acids, arranged in an order specified by DNA sequences. The cell strings together a sequence of amino acids based on a DNA code comprised of only four nucleobases: A (adenine), T (thymine), G (guanine) and C (cytosine) . The central steps in this process are illustrated in Figure 1 . Figure 1 : DNA specifies the sequence of a protein via the genetic code. The first step involves making an RNA version of the DNA’s sequence. This process is called transcription and uses one of the DNA strands to make a complementary copy that becomes a messenger RNA (mRNA) . The mRNA strand also has four bases: three of them (A, G, and C) are the same as DNA but mRNA contains uracil (U) instead of thymine (T). The term “complementary” refers to the fact that a specific base within the DNA strand (e.g., A) always pairs with a specific base within the mRNA strand (e.g., U). These base pairing rules ensure that the sequence of bases in mRNA is a faithful replica (although not an exact copy) of the sequence of bases in its parent DNA. Figure 2 on the following page illustrates the structures of DNA and mRNA side-by-side for comparison.

Laboratory 6: Cracking the Genetic Code 4 There are twenty different types of amino acids commonly found within proteins and they share a general structure shown in Figure 3(a) . Each type of amino acids differs in the chemical composition of its sidechain , which is represented as R . These sidechains have varying structures and properties – some sidechains are simple hydrocarbons whereas other contain various functional groups. Figure 3(b) shows three different amino acids and highlights their sidechain structures. (a) (b) Figure 3: (a) General structure of an amino acid. (b) The three amino acids shown differ in the chemical structures of their sidechains (highlighted in dashed boxes).

Laboratory 6: Cracking the Genetic Code 5 Table 1 lists all twenty amino acids according to their full name and three-letter symbol in alphabetical order. Although many of the names will be unfamiliar, they all represent variations on the chemical structure shown in Figure 3(a) . It is not necessary to memorize this list and the table can be used for reference during the laboratory exercise. Table 1 : Alphabetical listing of the 20 common amino acids 3 LETTER SYMBOL FULL NAME Ala alanine Arg arginine Asn asparagine Asp aspartic acid Cys cysteine Gln glutamine Glu glutamic acid Gly glycine His histidine Ile isoleucine Leu leucine Lys lysine Met methionine Phe phenylalanine Pro proline Ser serine Thr threonine Trp tryptophan Tyr tyrosine Val valine In order for scientists to “crack” the genetic code, they had to determine the rules that relate a particular set of three mRNA bases (a codon) to a particular amino acid. This was achieved through systematically making a variety of different mRNA sequences and examining what sequence of amino acids they produced via translation. In living cells this process occurs within the ribosome , a complex molecular factory for making proteins. With the aid of transfer RNA (tRNA) , the ribosome is able to form the amino acid chains that make up the structure of proteins. Scientists devised a way to mimic this process in the laboratory using “extracts” from bacterial cells that contains the necessary protein-synthesizing components. This enables translation to be carried out in a controlled environment and controlled without interference from other cellular processes. The mechanism behind protein synthesis in the cell is shown in Figure 4 on the following page.

Laboratory 6: Cracking the Genetic Code 7 COVER SHEET LABORATORY 6: CRACKING THE GENETIC CODE HUMAN GENETICS – CORE-UA 303 – Professor Fitch Name: ______________________________________________ Date: ____________________ Lab Partner Name(s): ______________________________________________ ______________________________________________ Laboratory Instructor: _________________________________ Section: __________________

Laboratory 6: Cracking the Genetic Code 8 CLEAN-UP CHECKLIST It is essential that you clean up properly after the experiments are complete.  Please ensure that the laptops are left with the Expasy Homepage open. NOTE : Check with your lab instructor that your bench is suitably clean before leaving the lab. Complete clean-up procedures are worth 5 points towards your lab score. Lab Instructor Initials _______________ GRADING Attendance: __________ / 5 Quiz: __________ / 10 Lab Project: __________ / 30 Clean Up: __________ / 5 Total: __________ / 50 *For TA use only*

Laboratory 6: Cracking the Genetic Code 10 Reproduce this famous experiment using a computer simulation First, make sure the Translate web site works for you: 1. Go to the following URL: https://web.expasy.org/translate/ 2. Use the following settings: o For "Output format", select "Verbose: Met, Stop, spaces between residues". o For "DNA strands", select "forward" only (i.e., deselect "reverse"). o For "Genetic codes", select the default "Standard". 3. Try entering a poly-U sequence containing at least 9 Uracil nucleotides (UUUUUUUUUUU) into the text box under the header "DNA or RNA sequence 4. After entering your RNA sequence, click the TRANSLATE button. 5. The RNA sequence that you typed into the box is automatically reverse-transcribed to a DNA sequence, but that is okay! Just look at the Results of translation, which shows the results of different "reading frames".

Laboratory 6: Cracking the Genetic Code 11 Question 2 [30 Points]: (a) What is a "reading frame"? Why are there three of them, labeled 1-3? (b) This poly-U sequence actually represents the first experiment by Nirenberg and Matthaei. What do you deduce is the amino acid that corresponds to the triplet codon UUU? (c) Design your simulation "experiment" to test what amino acid corresponds to AAA, CCC and GGG. What is your design, results and conclusions?

Laboratory 6: Cracking the Genetic Code 13 Khorana, Holley and Nirenberg were awarded the Nobel Prize in Physiology or Medicine in 1968 for their work leading to the elucidation of the genetic code. Now you might have an idea for why they deserved it!