Molecular GeneticsMolecular Genetics

The structure and packaging of The structure and packaging of DNADNA

ReplicationReplicationProtein SynthesisProtein Synthesis

What is a Chromosome?What is a Chromosome?Once chromosomes were identifiedOnce chromosomes were identified

Question #1 which part, the DNA or the Question #1 which part, the DNA or the protein, carries the genetic information?protein, carries the genetic information?

1952, Alfred Hershey and Martha Chase 1952, Alfred Hershey and Martha Chase used bacteriophages to determine it was used bacteriophages to determine it was the DNAthe DNA

Question #2 what was the structure of Question #2 what was the structure of DNADNAJames Watson and Francis Crick solved James Watson and Francis Crick solved the puzzle with the aid of X-ray the puzzle with the aid of X-ray crystallography done by Rosalind crystallography done by Rosalind Franklin and Maurice WilkinsFranklin and Maurice Wilkins

Fig. 16-3Fig. 16-3

Bacterial cell

Phage head

Tail sheath

Tail fiber

DNA

100

nm

Watson and Crick’s modelWatson and Crick’s model

3 major features3 major features1. double helix1. double helix2. nitrogenous bases of adenine (A), 2. nitrogenous bases of adenine (A), thymine (T), guanine (G), and cytosine (C) thymine (T), guanine (G), and cytosine (C) and the base paring of A-T and G-Cand the base paring of A-T and G-C3. antiparallel strands, one runs 5’ to 3’ 3. antiparallel strands, one runs 5’ to 3’ the opposite strand runs 3’ to 5’, the the opposite strand runs 3’ to 5’, the nucleotide monomers are flipped relative nucleotide monomers are flipped relative to each other and held together with H-to each other and held together with H-bondbond

Fig. 16-1Fig. 16-1

Fig. 16-6Fig. 16-6

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

Fig. 16-UN1Fig. 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

ReplicationReplication The process of making DNA from an existing The process of making DNA from an existing

DNA, result two daughter molecules composed DNA, result two daughter molecules composed of one new strand and one old strand from the of one new strand and one old strand from the original DNA.original DNA.

6 points of replication:6 points of replication:1.1. Origins of replication-Origins of replication- site where it begins site where it begins2.2. Initiation proteins, Initiation proteins, DNA helicaseDNA helicase, bind to the , bind to the

origin of replication and separate the two origin of replication and separate the two strands, forming a strands, forming a replication bubblereplication bubble. . Replication proceeds in both directions Replication proceeds in both directions expanding the bubble until complete.expanding the bubble until complete.

Replication ContinuedReplication Continued

3. 3. DNA polymeraseDNA polymerase catalyzes the elongation catalyzes the elongation of the new DNA at the replication forkof the new DNA at the replication fork

4. DNA polymerase adds nucleotides to the 4. DNA polymerase adds nucleotides to the growing chain one by one, working in a 5’ growing chain one by one, working in a 5’ to 3’ direction on the to 3’ direction on the leading strandleading strand, , pairing the bases to their complementpairing the bases to their complement

5. The 3’ to 5’ or 5. The 3’ to 5’ or lagging strandlagging strand is copied in is copied in segments called segments called Okazaki fragmentsOkazaki fragments

6. 6. DNA ligaseDNA ligase is the enzyme that puts them is the enzyme that puts them all together – “zips it up”all together – “zips it up”

Other factors involvedOther factors involved Base pairing rules are checked and double Base pairing rules are checked and double

checkedchecked Mismatch repair and Nucleotide excision repair, Mismatch repair and Nucleotide excision repair,

enzymes called enzymes called nucleasesnucleases remove and then remove and then replace the mistakes made during replicationreplace the mistakes made during replication

During replication the DNA polymerase is limited During replication the DNA polymerase is limited at the 5’ end, to ensure all essential nucleotide at the 5’ end, to ensure all essential nucleotide units are replicated there is a short repetitive units are replicated there is a short repetitive nucleotide sequence that does not contain genes nucleotide sequence that does not contain genes called the called the telomere.telomere. Each time the DNA replicates Each time the DNA replicates for Mitosis a small portion of this telomere is for Mitosis a small portion of this telomere is removed. removed. TelomeraseTelomerase is the enzyme responsible is the enzyme responsible for regenerating this segment. Research today is for regenerating this segment. Research today is looking at this enzyme and its role in cell aging.looking at this enzyme and its role in cell aging.

Eukaryotic and Prokaryotic DNAEukaryotic and Prokaryotic DNA A bacterial chromosome is one double-A bacterial chromosome is one double-

stranded, circular DNA molecule stranded, circular DNA molecule associated with a small amount of proteinassociated with a small amount of protein

Eukaryotic chromosomes are linear DNA Eukaryotic chromosomes are linear DNA molecules associated with large amounts molecules associated with large amounts of protein.of protein.• Chromatin: 4 levels of packingChromatin: 4 levels of packing

DNA wrapped around histones, like beads on a string DNA wrapped around histones, like beads on a string called a called a nucleosomenucleosome

Nucleosomes fold to form a Nucleosomes fold to form a 30nm fiber30nm fiber Further folding results in Further folding results in looped domainslooped domains of 300nm of 300nm Looped domains fold to form a Looped domains fold to form a metaphase metaphase

chromosomechromosome

Fig. 16-21aFig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome(10 nm in diameter)

Histones Histone tailH1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

States of ChromatinStates of Chromatin

During interphase chromosomes are During interphase chromosomes are in different statesin different states• EuchromatinEuchromatin, very loose and highly , very loose and highly

accessible to transcription enzymesaccessible to transcription enzymes• HeterochromatinHeterochromatin, more condensed and , more condensed and

not easily accessible to transcription not easily accessible to transcription enzymes. Barr bodies are enzymes. Barr bodies are heterochromatinheterochromatin

Protein Synthesis the basicsProtein Synthesis the basics

Gene expression is the process by which Gene expression is the process by which DNA directs the synthesis of proteinsDNA directs the synthesis of proteins

One gene- one polypeptide hypothesis One gene- one polypeptide hypothesis states that each gene codes for a specific states that each gene codes for a specific polypeptidepolypeptide

The DNA that will be transcribed is called The DNA that will be transcribed is called the the template strandtemplate strand

The code for a polypeptide chain is written The code for a polypeptide chain is written as a series of three- nucleotide groups as a series of three- nucleotide groups called a called a triplet code.triplet code.

Protein Synthesis Cont’dProtein Synthesis Cont’d

During transcription, the mRNA, During transcription, the mRNA, complementary to the DNA strand, is complementary to the DNA strand, is constructed of base triplets called constructed of base triplets called codons,codons, written in the 5’ to 3’ direction.written in the 5’ to 3’ direction.

There are 64 possible codons which code There are 64 possible codons which code for the 20 different amino acid subunits for for the 20 different amino acid subunits for proteins, the code is redundant. See chartproteins, the code is redundant. See chart

AUG is universal start codon, and UGA, AUG is universal start codon, and UGA, UAA, and UAG are stop codonsUAA, and UAG are stop codons

TranscriptionTranscription RNA polymerase separates the strands exposing RNA polymerase separates the strands exposing

the the transcription unittranscription unit (gene) and connects the (gene) and connects the RNA nucleotides to their complement on the DNA RNA nucleotides to their complement on the DNA template strandtemplate strand. The DNA sequence in which the . The DNA sequence in which the RNA polymerase attaches is called the RNA polymerase attaches is called the promoterpromoter

RNA polymerase adds the RNA nucleotides to the RNA polymerase adds the RNA nucleotides to the 3’ end and the RNA elongates (3’ end and the RNA elongates (uracil uracil replaces replaces thymine in the code so that A- U) thymine in the code so that A- U)

This continues until it reaches the This continues until it reaches the terminatorterminator, the , the stop sequence on the DNA strand, this releases stop sequence on the DNA strand, this releases the RNA transcript and the RNA polymerase the RNA transcript and the RNA polymerase detaches.detaches.

summarysummary 3 main stages of transcription3 main stages of transcription

1. Initiation1. Initiation2. Elongation2. Elongation3. Termination3. Termination

Modification of the RNA transcription sequenceModification of the RNA transcription sequence• 5’ cap and poly-A tail5’ cap and poly-A tail facilitate the export of the RNA out of facilitate the export of the RNA out of

the nucleusthe nucleus• Ribozymes Ribozymes (small nuclear RNA or snRNA) splice the mRNA (small nuclear RNA or snRNA) splice the mRNA

removing removing intronsintrons and then splice together the remaining and then splice together the remaining exonsexons

• New understanding of gene expression- 25,000 genes and New understanding of gene expression- 25,000 genes and 100, 000 polypeptides. These are the result of alternative 100, 000 polypeptides. These are the result of alternative RNA splicing allows for different combinations of exons, and RNA splicing allows for different combinations of exons, and more than one polypeptide per genemore than one polypeptide per gene

Translation, mRNA- polypeptideTranslation, mRNA- polypeptide

Two more types of RNA, transfer or Two more types of RNA, transfer or tRNAtRNA and and ribosomal or ribosomal or rRNArRNA

tRNA transfers amino acids (a.a.) from the cell’s tRNA transfers amino acids (a.a.) from the cell’s cytoplasm to a ribosome. The ribosome accepts the cytoplasm to a ribosome. The ribosome accepts the a.a. and incorporates it into a growing polypeptide a.a. and incorporates it into a growing polypeptide chain.chain.

Each tRNA is specific for a particular a.a., at one Each tRNA is specific for a particular a.a., at one end it loosely binds the a.a., and at the other end it end it loosely binds the a.a., and at the other end it has a nucleotide triplet called an has a nucleotide triplet called an anticodon. anticodon. The The anticodon pairs with the complementary mRNA anticodon pairs with the complementary mRNA codoncodon

rRNA forms the ribosome which is made of two rRNA forms the ribosome which is made of two subunits, one small subunit and one large subunit.subunits, one small subunit and one large subunit.

TranslationTranslation The mRNA is read codon by codon starting with the The mRNA is read codon by codon starting with the

initiating codon AUG, and one amino acid is added for each initiating codon AUG, and one amino acid is added for each codon translated by the ribosome.codon translated by the ribosome.

The ribosome together with initiation factors (proteins that The ribosome together with initiation factors (proteins that aid in the translation process) make up the aid in the translation process) make up the Translation Translation initiation complexinitiation complex the two ribosomal subunits come the two ribosomal subunits come together during initiationtogether 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 A site for the tRNA that holds the a.a. that will be added to the

polypeptide nextpolypeptide next• E site for the tRNA that will be exiting now that it has passed 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.on its polypeptide to the tRNA sitting in the P site.

TranslationTranslation Elongation has 3 stepsElongation 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 Peptide bond formation, the a.a. attached to

the tRNA sitting in site A is bonded to the a.a. the tRNA sitting in site A is bonded to the a.a. sequence sitting in site Psequence sitting in site P

• Translocation, when the polypeptide bond Translocation, when the polypeptide bond forms the tRNA in site P shifts to site E, and forms the tRNA in site P shifts to site E, and tRNA is site A shifts to site P now that it is tRNA is site A shifts to site P now that it is vacant…vacant…

Termination is when a stop codon in the Termination is when a stop codon in the mRNA is reached, the polypeptide is freed mRNA is reached, the polypeptide is freed from the ribosomefrom the ribosome

Protein structureProtein structure Signal Peptide-Signal Peptide- the first 20 a.a. on the the first 20 a.a. on the

leading end of a polypeptide direct the leading end of a polypeptide direct the protein to its final destination (~zip code)protein to its final destination (~zip code)

Mutagens, substances or forces that Mutagens, substances or forces that interact with DNA in ways that cause interact with DNA in ways that cause mutations. X-rays, and other radiation, mutations. X-rays, and other radiation, chemicals (carcinogens) chemicals (carcinogens)

Mutations, alterations in the genetic Mutations, alterations in the genetic materialmaterialPoint mutations alterations of just one base pair Point mutations alterations of just one base pair

in a gene, two typesin a gene, two types Base-pair substitution (missense and nonsense)Base-pair substitution (missense and nonsense) Insertions/deletions cause a frame shiftInsertions/deletions cause a frame shift

Define Gene…Define Gene…

A gene is a region of DNA that can be A gene is a region of DNA that can be expressed to produce a final functional expressed to produce a final functional product that is either a polypeptide or an product that is either a polypeptide or an RNA molecule (tRNA, rRNA, snRNA)RNA molecule (tRNA, rRNA, snRNA)

It is the polypeptides that form the It is the polypeptides that form the proteins, which, in turn, bring about an proteins, which, in turn, bring about an organism's observable phenotype.organism's observable phenotype.

A given type of cell expresses only a A given type of cell expresses only a subset of its genes. This is an essential subset of its genes. This is an essential feature of multicellular organisms. Gene feature of multicellular organisms. Gene expression is precisely regulated.expression is precisely regulated.