Molecular Genetics The structure and packaging of DNA Replication Protein Synthesis

What is a Chromosome? Once chromosomes were identified Question #1 which part, the DNA or the protein, carries the genetic information? 1952, Alfred Hershey and Martha Chase used bacteriophages to determine it was the DNA Question #2 what was the structure of DNA James Watson and Francis Crick solved the puzzle with the aid of X-ray crystallography done by Rosalind Franklin and Maurice Wilkins

Fig Bacterial cell Phage head Tail sheath Tail fiber DNA 100 nm

Watson and Crick’s model 3 major features 3 major features 1. double helix 2. nitrogenous bases of adenine (A), thymine (T), guanine (G), and cytosine (C) and the base paring of A-T and G-C 3. antiparallel strands, one runs 5’ to 3’ the opposite strand runs 3’ to 5’, the nucleotide monomers are flipped relative to each other and held together with H- bond

Fig. 16-1

Fig (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

Fig. 16-UN1 Purine + purine: too wide Purine + pyrimidine: width consistent with X-ray data Pyrimidine + pyrimidine: too narrow The Watson-Crick model explains Chargoff’s rules: in any organism the amount of A = T, and the amount of G = C

Replication The process of making DNA from an existing DNA, result two daughter molecules composed of one new strand and one old strand from the original DNA. The process of making DNA from an existing DNA, result two daughter molecules composed of one new strand and one old strand from the original DNA. 6 points of replication: 6 points of replication: 1. Origins of replication- site where it begins 2. Initiation proteins, DNA helicase, bind to the origin of replication and separate the two strands, forming a replication bubble. Replication proceeds in both directions expanding the bubble until complete.

Replication Continued 3. DNA polymerase catalyzes the elongation of the new DNA at the replication fork 4. DNA polymerase adds nucleotides to the growing chain one by one, working in a 5’ to 3’ direction on the leading strand, pairing the bases to their complement 5. The 3’ to 5’ or lagging strand is copied in segments called Okazaki fragments 6. DNA ligase is the enzyme that puts them all together – “zips it up”

Other factors involved Base pairing rules are checked and double checked Base pairing rules are checked and double checked Mismatch repair and Nucleotide excision repair, enzymes called nucleases remove and then replace the mistakes made during replication Mismatch repair and Nucleotide excision repair, enzymes called nucleases remove and then replace the mistakes made during replication During replication the DNA polymerase is limited at the 5’ end, to ensure all essential nucleotide units are replicated there is a short repetitive nucleotide sequence that does not contain genes called the telomere. Each time the DNA replicates for Mitosis a small portion of this telomere is removed. Telomerase is the enzyme responsible for regenerating this segment. Research today is looking at this enzyme and its role in cell aging. During replication the DNA polymerase is limited at the 5’ end, to ensure all essential nucleotide units are replicated there is a short repetitive nucleotide sequence that does not contain genes called the telomere. Each time the DNA replicates for Mitosis a small portion of this telomere is removed. Telomerase is the enzyme responsible for regenerating this segment. Research today is looking at this enzyme and its role in cell aging.

Eukaryotic and Prokaryotic DNA A bacterial chromosome is one double- stranded, circular DNA molecule associated with a small amount of protein A bacterial chromosome is one double- stranded, circular DNA molecule associated with a small amount of protein Eukaryotic chromosomes are linear DNA molecules associated with large amounts of protein. Eukaryotic chromosomes are linear DNA molecules associated with large amounts of protein. Chromatin: 4 levels of packing Chromatin: 4 levels of packing DNA wrapped around histones, like beads on a string called a nucleosome DNA wrapped around histones, like beads on a string called a nucleosome Nucleosomes fold to form a 30nm fiber Nucleosomes fold to form a 30nm fiber Further folding results in looped domains of 300nm Further folding results in looped domains of 300nm Looped domains fold to form a metaphase chromosome Looped domains fold to form a metaphase chromosome

Fig a DNA double helix (2 nm in diameter) Nucleosome (10 nm in diameter) Histones Histone tail H1 DNA, the double helixHistones Nucleosomes, or “beads on a string” (10-nm fiber)

States of Chromatin During interphase chromosomes are in different states During interphase chromosomes are in different states Euchromatin, very loose and highly accessible to transcription enzymesEuchromatin, very loose and highly accessible to transcription enzymes Heterochromatin, more condensed and not easily accessible to transcription enzymes. Barr bodies are heterochromatinHeterochromatin, more condensed and not easily accessible to transcription enzymes. Barr bodies are heterochromatin

Protein Synthesis the basics Gene expression is the process by which DNA directs the synthesis of proteins Gene expression is the process by which DNA directs the synthesis of proteins One gene- one polypeptide hypothesis states that each gene codes for a specific polypeptide One gene- one polypeptide hypothesis states that each gene codes for a specific polypeptide The DNA that will be transcribed is called the template strand The DNA that will be transcribed is called the template strand The code for a polypeptide chain is written as a series of three- nucleotide groups called a triplet code. The code for a polypeptide chain is written as a series of three- nucleotide groups called a triplet code.

Protein Synthesis Cont’d During transcription, the mRNA, complementary to the DNA strand, is constructed of base triplets called codons, written in the 5’ to 3’ direction. During transcription, the mRNA, complementary to the DNA strand, is constructed of base triplets called codons, written in the 5’ to 3’ direction. There are 64 possible codons which code for the 20 different amino acid subunits for proteins, the code is redundant. See chart There are 64 possible codons which code for the 20 different amino acid subunits for proteins, the code is redundant. See chart AUG is universal start codon, and UGA, UAA, and UAG are stop codons AUG is universal start codon, and UGA, UAA, and UAG are stop codons

Transcription RNA polymerase separates the strands exposing the transcription unit (gene) and connects the RNA nucleotides to their complement on the DNA template strand. The DNA sequence in which the RNA polymerase attaches is called the promoter RNA polymerase separates the strands exposing the transcription unit (gene) and connects the RNA nucleotides to their complement on the DNA template strand. The DNA sequence in which the RNA polymerase attaches is called the promoter RNA polymerase adds the RNA nucleotides to the 3’ end and the RNA elongates (uracil replaces thymine in the code so that A- U) RNA polymerase adds the RNA nucleotides to the 3’ end and the RNA elongates (uracil replaces thymine in the code so that A- U) This continues until it reaches the terminator, the stop sequence on the DNA strand, this releases the RNA transcript and the RNA polymerase detaches. This continues until it reaches the terminator, the stop sequence on the DNA strand, this releases the RNA transcript and the RNA polymerase detaches.

summary 3 main stages of transcription 3 main stages of transcription 1. Initiation 2. Elongation 3. Termination Modification of the RNA transcription sequence Modification of the RNA transcription sequence 5’ cap and poly-A tail facilitate the export of the RNA out of the nucleus5’ cap and poly-A tail facilitate the export of the RNA out of the nucleus Ribozymes (small nuclear RNA or snRNA) splice the mRNA removing introns and then splice together the remaining exonsRibozymes (small nuclear RNA or snRNA) splice the mRNA removing introns and then splice together the remaining exons New understanding of gene expression- 25,000 genes and 100, 000 polypeptides. These are the result of alternative RNA splicing allows for different combinations of exons, and more than one polypeptide per geneNew understanding of gene expression- 25,000 genes and 100, 000 polypeptides. These are the result of alternative RNA splicing allows for different combinations of exons, and more than one polypeptide per gene

Translation, mRNA- polypeptide Two more types of RNA, transfer or tRNA and ribosomal or rRNA Two more types of RNA, transfer or tRNA and ribosomal or rRNA tRNA transfers amino acids (a.a.) from the cell’s cytoplasm to a ribosome. The ribosome accepts the a.a. and incorporates it into a growing polypeptide chain. tRNA transfers amino acids (a.a.) from the cell’s cytoplasm to a ribosome. The ribosome accepts the a.a. and incorporates it into a growing polypeptide chain. Each tRNA is specific for a particular a.a., at one end it loosely binds the a.a., and at the other end it has a nucleotide triplet called an anticodon. The anticodon pairs with the complementary mRNA codon Each tRNA is specific for a particular a.a., at one end it loosely binds the a.a., and at the other end it has a nucleotide triplet called an anticodon. The anticodon pairs with the complementary mRNA codon rRNA forms the ribosome which is made of two subunits, one small subunit and one large subunit. rRNA forms the ribosome which is made of two subunits, one small subunit and one large subunit.

Translation The mRNA is read codon by codon starting with the initiating codon AUG, and one amino acid is added for each codon translated by the ribosome. The mRNA is read codon by codon starting with the initiating codon AUG, and one amino acid is added for each codon translated by the ribosome. The ribosome together with initiation factors (proteins that aid in the translation process) make up the Translation initiation complex the two ribosomal subunits come together during initiation The ribosome together with initiation factors (proteins that aid in the translation process) make up the Translation initiation complex the two ribosomal subunits come together during initiation The ribosome has 3 binding sites for the tRNA. The ribosome has 3 binding sites for the tRNA. P site for the tRNA that holds the polypeptideP site for the tRNA that holds the polypeptide A site for the tRNA that holds the a.a. that will be added to the polypeptide nextA site for the tRNA that holds the a.a. that will be added to the polypeptide next E site for the tRNA that will be exiting now that it has passed on its polypeptide to the tRNA sitting in the P site.E site for the tRNA that will be exiting now that it has passed on its polypeptide to the tRNA sitting in the P site.

Translation Elongation has 3 steps Elongation has 3 steps Codon recognition, codon in site A is matchedCodon recognition, codon in site A is matched Peptide bond formation, the a.a. attached to the tRNA sitting in site A is bonded to the a.a. sequence sitting in site PPeptide bond formation, the a.a. attached to the tRNA sitting in site A is bonded to the a.a. sequence sitting in site P Translocation, when the polypeptide bond forms the tRNA in site P shifts to site E, and tRNA is site A shifts to site P now that it is vacant…Translocation, when the polypeptide bond forms the tRNA in site P shifts to site E, and tRNA is site A shifts to site P now that it is vacant… Termination is when a stop codon in the mRNA is reached, the polypeptide is freed from the ribosome Termination is when a stop codon in the mRNA is reached, the polypeptide is freed from the ribosome

Protein structure Signal Peptide- the first 20 a.a. on the leading end of a polypeptide direct the protein to its final destination (~zip code) Signal Peptide- the first 20 a.a. on the leading end of a polypeptide direct the protein to its final destination (~zip code) Mutagens, substances or forces that interact with DNA in ways that cause mutations. X-rays, and other radiation, chemicals (carcinogens) Mutagens, substances or forces that interact with DNA in ways that cause mutations. X-rays, and other radiation, chemicals (carcinogens) Mutations, alterations in the genetic material Mutations, alterations in the genetic material Point mutations alterations of just one base pair in a gene, two types Base-pair substitution (missense and nonsense) Base-pair substitution (missense and nonsense) Insertions/deletions cause a frame shift Insertions/deletions cause a frame shift

Define Gene… A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule (tRNA, rRNA, snRNA) A gene is a region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule (tRNA, rRNA, snRNA) It is the polypeptides that form the proteins, which, in turn, bring about an organism's observable phenotype. It is the polypeptides that form the proteins, which, in turn, bring about an organism's observable phenotype. A given type of cell expresses only a subset of its genes. This is an essential feature of multicellular organisms. Gene expression is precisely regulated. A given type of cell expresses only a subset of its genes. This is an essential feature of multicellular organisms. Gene expression is precisely regulated.