I. DNA as Genetic Material Frederick Griffith Avery, McCarty, MacLeod Hershey and Chase Chargaff Pauling Wilkins and Franklin Watson and Crick

Watson and Crick determined that there are 2 H bonds between A and T and 3 H bonds between C and G

ATGTCGATGTCG TACAGCTACAGC ATGTCGATGTCG TACAGCTACAGC TACAGCTACAGC ATGTCGATGTCG +

Semi-Conservative Theory of Replication DNA always replicates in a 5’ to 3’ direction The 5’ and 3’ must always be inverted next to one another.

DNA Replication begins at a special site called the origins of replication. A specific sequence of nucleotides required in initiate replication. DNA creates a replication fork that opens at the initiation point and creates a replication bubble.

Strand separation is achieved by: Helicases = enzymes that unwind the parental double helix Single strand binding proteins = keep the strands apart and stabilize the unwound DNA strand

DNA Polymerase = catalyze the synthesis of a new DNA strand Strand can only grow in a 5’  3’ direction Nucleoside triphosphate = provides energy to form new bonds (two phosphates are lost = endergonic rex)

Leading Strand = synthesized as a single unit in the 5’  3’ direction towards the replication fork Lagging Strand = discontinuously synthesized against the overall direction of replication OKAZAKI FRAGMENTS

Lagging Strand = produces short segments in order to continue to produce 5’  3’ ~ 1000 – 2000 nucleotides long in bacteria ~ nucleotides long in eukaryotes Fragments are joined together by DNA LIGASE

Primer = short RNA segment complementary to the DNA strand that starts DNA replication * Polymerized by PRIMASE * One primer is required for the leading strand * Many primers are required for the lagging strand

PROOFREADING: initial base pairing errors are rare (~1 in 10,000) base pairing is double checked by DNA polymerase which compares added nucleotides against its template *errors are removed and replaced

Genes to Proteins: Garrod= proposes relationship between genes and proteins Beadle and Ephrussi= studied the link between eye color and enzyme production in Drosophila Beadle and Tatum= demonstrated link between genes in enzymes in bread mold (Neurospora crassa)

Protein Synthesis involves: Transcription = synthesis of a mRNA strand using the DNA template Translation = synthesis of a polypeptide under the direction of the mRNA strand

RNA Binding and Initiation: * RNA polymerases bind to DNA at the promoter region * Most eukaryotes also contain transcription factors = specific instructions for coding the mRNA strand

TATA box = a short nucleotide sequence of T’s and A’s right before the promoter region to help the RNA polymerase to recognize the promoter region

Elongation of the RNA Strand: As the RNA polymerase II moves along the DNA strand: the helix untwists and opens exposing about 10 base pairs at a time it links to RNA nucleotides at the 3’ end and the RNA strand grows in a 5’ to 3’ direction

Termination of Transcription: * Transcription continues until RNA polymerase reaches a terminator sequence on the DNA * Acts as a STOP sign for the RNA polymerase transcription

TRANSLATION: tRNA acts as interpreter between two forms of infomation tRNA aligns the appropriate amino acids to form a new poly- peptide

tRNA molecules: transfer amino acids from the cytoplasm’s amino acid pool to a ribosome must recognize the correct codons in mRNA are specific for only one amino acid have one end attached to a specific amino acid the other end attaches to an mRNA codon by pairing with its anticodon

ANTICODON: a nucleotide triplet in the tRNA that base pairs with a complementary nucleotide triplet codon in mRNA

CODONS: a 3 nucleotide sequence in mRNA that specifies which amino acid will be added *there are 64 codons and 20 amino acids so there is redundancy = two or more codons code for the same amino acid * There are 45 distinct types of tRNA so each tRNA molecule recognizes 2-3 mRNA codons specifying the same amino acid

Since there is redundancy: WOBBLE = the use of inosine (I) a modified base in the third position (the wobble position) CCI codes for GGU, GGA, GGC and GGG (which all code for glycine)

Aminoacyl-tRNA Synthetase = a type of enzyme that catalyzes the attachment of an amino acid to its tRNA *each of the 20 amino acids has its own specific aminoacyl-tRAN sythetase * Requires an endergonic reaction (ATP driven) that attaches an amino acid to its tRNA by:

1. Activation of amino acid by AMP: the synthetase’s active site binds to an amino acid and ATP  the ATP loses two phosphates and attaches to the amino acid as AMP (adenosine monophosphate) 2. Attachment of the amino acid to tRNA: the appropriate tRNA covalently bonds to the amino acid, displacing AMP from the enzyme’s active site

RIBOSOMES: coordinate pairing of tRNA anitcodons to mRNA codons * Have two subunits (small and large) which are separated when not in- volved in protein synthesis * When protein synthesis begins, a small subunit and a large subunit come together and bind to the mRNA and the tRNA

P SITE = holds the tRNA carrying the growing poly- peptide chain A SITE = holds the next amino acid to be added

Initiation: brings together the mRNA, the first amino acid attached to the tRNA, and the two ribosomal units * Requires one GTP molecule to help stabilize the attachment of the large ribosomal unit ( functional ribosome)

ELONGATION: proteins add amino acids one by one to the initial amino acid Codon Recognition: mRNA codon in A site forms H-bonds with the anticodon of entering tRNA carrying new amino acids (GTP provides the energy) Peptide Bond Formation: polypeptide transferase catalyzes formation of a peptide bond between polypeptide at P and a new amino acid at A

Translocation: the tRNA at P releases from the ribosome and tRNA at A is translocated to P codon and anticodon remain bonded, so mRNA and tRNA move as a unit bringing the next codon into the A site mRNA moves through the ribosome only in the 5’ to 3’ direction GTP provides the energy for each translocation

Termination: end point of translation when the stop codon is reached when the stop codon is reached, ribosomes at the A site binds to a release factor instead of an amino- acyl tRNA synthetase * Causes the ribosomal subunits to dissociate from the mRNA strand and separate back into small and large subunits

Polyribosomes: cluster of ribosomes simultaneously translating an mRNA molecule

*Transcription occurs in the nucleus * Translation occurs in the cytoplasm * In eukaryotes, the RNA is processed, so that it is not damaged while moving from the nucleus to the cytoplasm.

RNA Processing: 5’ Cap: GTP added to 5’ end of mRNA after transcription begins to protect it from hydrolytic enzymes Poly A Tail: sequence of a 100+ adenine nucleotides added to the 3’ end of the mRNA before it leaves the nucleus to prevent degradation in the cytoplasm

RNA Splicing: process of removing introns and joining exons to form complete mRNA strand because INTRONS: noncoding sequences in DNA that are positioned between exons EXONS: coding sequences of a gene