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

ATGTCG

TACAGC

ATGTCG

TACAGC

TACAGC

ATGTCG

+

Semi-Conservative Theory of ReplicationDNA always replicates in a 5’ to 3’ directionThe 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 forkLagging 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~ 100-200 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 proteinsBeadle and Ephrussi= studied the link between eye color and enzyme production in DrosophilaBeadle 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 acidCodon 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