Brandy Hucke (Introduction) Dustin Ahlschwede (Method) Joshua Theobald (Materials) Ashley Betke (Results) Justin Byers (Discussion) Joseph Peters (Critique)

Why was this research of such interest? The genetic code is essentially the code of life, and the possible applications resulting from cracking that code enables researchers to create strands of DNA and RNA to produce proteins necessary for maintaining life at a higher standard. For example, this process can create insulin (to treat diabetic patients) as well as different coagulation factors used to treat hemophilia patients.

Background Information: DNA -Proteins are comprised of amino acids. -There are 20 different amino acids. -The genes that encode amino acids are found in DNA. -DNA is comprised of 4 nucleotides: Adenine, Thymine, Cytosine and Guanine. -Protein synthesis is the process by which DNA encodes for the production of amino acids and proteins. The question now is how does DNA direct protein synthesis?

Marshall Nirenberg, the scientist that deciphered the genetic code in 1961

-tRNA occurs in two separate forms. - “ Charged ” tRNA holds a covalent bond with a single amino acid. - “ Uncharged ” tRNA has no attached amino acid. -After mRNA is bonded with amino-acylated tRNA, bonds form between the amino acids. -Incoming tRNA continues to bind with mRNA, while amino acids bond, thus elongating the chain. -How does the mRNA direct protein synthesis? -Phenylalanie charged tRNA combined with ribosomes and polyuridylic acid (ployU) in an incubator produces phenylalanie pure peptides. -Thus, the mRNA codon for phenylalanie consists of nucleotides made up of the base uracil. -The mRNA codon for proline consists of nucleotides made up of the base cytadine. -The mRNA codon for lysine consists of nucleotides made up of the base adenine. -What is the minimum chain length needed for tRNA binding to ribosomes? -How does DNA direct protein synthesis? Background Information

Nirenberg developed a method to determine the answer to this question, which in turn enabled him to decipher which aminoacylated tRNA would bind to which mRNA codon; the key that unlocked the genetic code.

Determine length of mRNA required for tRNA  In vitro incubation of a known number of nucleotides  In vitro incubation of three components of protein synthesis  Recognition of the RNA complex with radioactivity  Separating the complex with the use of a nylon filter

Reasoning behind this choice  Simple  Precise  Multiple choices of use

The Break through This method not only accomplished determining the length of a codon, but could be used to test all the possible combinations of nucleotides

Materials used in Nirenberg’s experiment: ruptured E. coli bacteria cells (cell free system) synthetic RNA molecules 20 test tubes each filled with ‘soup’: a different amino acid, RNA template, ribosomes, binding factors, GTP 19 test tubes were “cold” and one was radioactively tagged so the scientists could watch the reaction nylon membranes

Their experiment required a cell-free system Nirenberg chose E. Coli bacteria cells

20 test tubes 19 test tubes were “cold” and one was radioactively tagged The “hot” amino acid would change every time they did the experiment.

The minimum chain length of RNA needed for tRNA recognition was reached. After trial and error it was found that trinucleotides, rather than dinucleotides, where the only kind where the tRNA binding to the ribosomes could be detected. This first started with UUU. It later led CCC and AAA to be tested. CCC recognized proline-charged tRNA. And AAA recognized lysine-charged tRNA.

After discovering this, it was concluded that these trinucleotides could recognize amino-acylated tRNAs. These results led to the possibility of testing all 64 possible combinations of trinucleotides that could essentially lead to cracking the genetic code.

What were the conclusions? The codons required for recognition of tRNA must be a trinucleotide. Trinucleotides effectively direct the proper recognition of amino-acylated tRNAs. What was the significance of this work? Nirenberg’s assay delivered a method to assign each specific amino acid to one or more trinucleotides. Twenty amino acids were assigned at least one trinucleotide, 61 in total. Three trinucleotides where determined to be “stop” codons.

Are there potential applications in research and medicine? By knowing the DNA sequence of a gene scientists can predict the amino acid sequence of the protein it will encode. More effective medicines This knowledge fuels research into genetic anomalies that cause disease and possible cures to such diseases. Research into gene manipulation is also occurring.

Critique the research. What is the next step if you were the one who finished the study?