GENE to PROTEIN • Garrod (1909) hypothesized that the symptoms of an inherited disease reflect a person’s inability to make a particular enzyme. • The breakthrough in demonstrating the relationship between genes and enzymes when Beadle and Tatum began to search for mutants of bread mold. They discovered that mutants differ from wild type in their nutritional needs. • Nutritional mutants are called auxotrophs. • Beadle and Tatum were able to demonstrate the relationship between genes and enzymes by studying mutant forms of bread mold. Each gene dictates the production of one enzyme. • Many genes are made from two or more polypeptide chains – therefore it is really one gene – one polypeptide. • Genes are typically hundreds or thousands of nucleotides long. The nucleic acids and proteins are written in two different languages. DNA must be converted to protein language. • This is accomplished through the processes of • Transcription • Translation Prokaryotes vs Eukaroytes • Prokaryotes lack nuclei and DNA is not segregated from ribosomes and transcription and translation occur in rapid succession. • Eukaryotes have a nuclear envelop envelop and that segregates transcription in the nucleus from translation that occurs in the cytoplasm. • The flow of information from a gene to a protein is based a a triplet code. • These three nucleotide “words” are codons. Cracking the Genetic Code • Nirenberg – 1961 NIH • First codon decipher was UUU • There are 64 codons • A codon codes for only 1 amino acid The genetic code must have evolved very early in the history of life because it is nearly universal among living organisms. Transcription • Transcription of messenger RNA from template DNA is catalyzed by RNA polymerase, which • Separate the two strands of DNA and link RNA nucleotides as they base-pair along the template • Add nucleotides to the 3’ end; thus mNRA grows in the 5’ 3’ direction • Transcription occurs in three stages • Polymerase binding and initiation • Elongation • Termination • In eukaryotes, RNA polymerase cannot recognize the promoter ( about 100 nucleotides long) without transcription factors. • RNA polymerase II cannot recognize the promotor site without binding to the TATA box ( a short sequence of nucleotides rich in A and T that is about 25 nucleotides upstream from the initiation site. RNA SPLICING • Introns – noncoding segments that lie between the coding segments • Exons -- coding segments that are expressed • Roberts and Sharp – independently found evidence for “split gene” and both received a Nobel Prize • RNA polymerase transcribes all the sequences and this is known as pre mRNA. This pre mRNA never leaves the nucleus. • The introns are removed and the “abridged” version of mRNA moves to the cytoplasm as the primary transcription. • There are signals for the RNA splicing at the ends of the introns. Particles called small nuclear ribonucleoproteins (snRNPs – “snurps”) begin the process. • Several snRNA molecules join to form a spliceosome ( about the size of a ribosome). These cut the introns and connect the exons. Ribozymes • RNA molecules that function as enzymes • Serve as a catalyst • Makes the following statement obsolete: “All biological catalyst are proteins.” What is the function of Introns and Splicing? • May control gene activity and may regulate passage of mRNA to cytoplasm • May have a role in the evolution of new proteins • Increases the probability of crossing-over • Split genes have a higher frequency of recombination. Translation RNA directed synthesis of a polypeptide • tRNA serves as the interpreter • The role of tRNA is to transfer amino acids from the “pool” in the cytoplasm to a ribosome. • The ribosome adds each amino acid to the growning end of a polypeptide chain ( 3’) Transfer RNA • Molecules of tRNA are not all identical • Each type of tRNA links a mRNA codon with a specific amino acid. • One end has an attachment site for amino acid and the anticodon is on the other end. • May be used repeatedly • Consists of a single RNA strand about 80 nucleotides long • Looks like a clover leaf • Loop protruding from L end is the anticodon and this binds to the mRNA • There are 45 tRNA molecules. ( Should be 61 if there was a tRNA for each codon) • Some tRNA can recognize 2 or more codons. • WOBBLE – Allows for the relaxation of the base-pairing rules • U can pair with A or G in the third position • The most versatile tRNA has the inosin (I). The I ( in the wobble position) can form hydrogen bonds with U, C or A Steps in Translation 1. Correct match between tRNA and a amino acid. The amino acid is bound to the tRNA by aminocyl tRAN synthetase. There are 20 of these – one for each amino acid 2. The codon-anticodon bonding. Ribosomes • 2 subunits • Large subunit • Small subunit • 60% of weight of ribosome is rRNA • 3 Binding sites for tRNA • P – holds the tRNA with the growing polypetide chain • A – hold the next amino acid to be added • E – exit site A single ribosome can make an average sized polypeptide in less than one minute. Usually a single mRNA is used to make many copies of a polypeptide simultaneously because several ribosomes work on translating at the same time. Polyribosome – string of ribosomes . Mutations Change in the genetic makeup • Point mutation – change in one or a few base-pairs in a single gene • If occurs in a gamete then the change is passed to the future. • If it has an adverse effect then it is known as a genetic disorder or hereditary disease Types of Point Mutations • Substitution 1. Misssense – still codes for an amino acid 2. Nonsense – does not code for an amino acid but changes to a code for STOP. This makes the chain too short and usually leads to nonfunctioning proteins. •Insertion or Deletion 1.Frameshift – not in a multiple of three • Spontaneous mutation are the result of errors in the DNA replication or repair. • Mutagens are chemical or physical agents that cause DNA to change. • AMES test – is a test for mutagenic activity of different chemicals.