Molecular GeneticsDNA Structure and

ReplicationChapter 11

* In the pasts, scientists argued about how genetic information was passed down and influcenced traits.

* Some scientists believed in "blending" - that the traits of two parents are mixed.  * This doesn't work when a white flower and a   purple flowered plant have a white flowered   offspring.

* Some scientists also believed that proteins were responsible for passing on traits, not DNA. They thought this because DNA molecules are so simple compared to proteins.

History of Molecular Genetics

Griffith & Avery * Took pathogenic bacteria (cause disease) and killed   them with heat.  * Mixed the dead bacteria with harmless bacteria.   * When they injected the mix into mice, it killed them. * The harmless bacteria must have taken up  something from the dead, harmful bacteria that  transformed the harmless bacteria * It was the DNA!

Current Thinking and the Experiments that Led to It

Current Thinking and the Experiments that Led to It

Avery, MacLeod, and McCarty * Tested what was being   transferred between the   bacteria...was it DNA?   RNA? Proteins? * Killed virulent bacteria * Subjected a sample to   

RNAase, one to Protease, one to DNAase * The samples treated with    RNAase and Protease

were still able to transform   harmless bacteria but   DNAase treated sample   could not * DNA must be the agent of    transformation

Current Thinking and theExperiments that Led to ItHershey & Chase

 * Radioactively labeled DNA and proteins in viruses.  * They allowed the viruses to infect cells * Waited to see whether it was the DNA or the protein that entered   the cells to infect them. * Found DNA to be radioactive and therefore the genetic material

Chargaff's Rule * Identified pairing rule of bases * Adenine = Thymine (2 H bonds) * Guanine = Cytosine (3 H bonds) Rosalind Franklin * took the first picture of a DNA molecule using   a technique called x-ray crystallography

Watson & Crick  * used Franklin's picture and Chargaff’s ratios and cutouts to determine DNA structure

Current Thinking and the Experiments that Led to It

DNA Structure videohttps://www.youtube.com/watch?v=C1CRrtkWwu0

Nucleotide - monomer of nucleic acids * DNA Nucleotide Structure - 3 Parts  1. Sugar (deoxyribose)

 2. Phosphate group  3. Nitrogenous Base - 4 Kinds  1. Adenine  2. Guanine

3. Cytosine  4. Thymine

 * Purines and Pyrimidines are ring structures associated with the Nitrogenous base  * Purines - 2 rings  *EX: A and G  * Pyrimidines - 1 rings

*EX: T and C

DNA Structure

DNA is a molecule made of 2 strands of nucleotides * Individual strands are made by connecting sugars and    phosphates of nucleotides with a phosphodiester bond (type of covalent) * Forms sugar - phosphate backbone * The two strands are connected by hydrogen bonds   between the bases * The two strands are wound together into a double helix.

DNA Structure

DNA StructureDirectionality of DNA strands * 3' end - end of each strand that has the

hydroxyl group off  the 3rd carbon of the sugar molecule * 5' end - the other end which has a Phosphate group off the 5th carbon of the sugar

molecule

* Anti-parallel- The 2 DNA strands run in

opposite directions to each other

DNA Organization * Chromosome - DNA wrapped around histone proteins (before cell division) * Chromatin - loose, uncoiled DNA (during Interphase) * Euchromatin - areas in chromosomes that are loosely opened so it  can be

transcribed * Heterochromatin - areas in chromosomes that are compacted and  generally not transcribed* Gene - A segment of DNA that codes for a specific trait  * Telomeres - repeated nucleotide sequences at the ends of chromosomes   * they do NOT code for proteins  * TTAGGG in humans -- it is repeated about 2500 times  * 50 -200 base pairs of telomeric DNA are lost after every cell division  * Cells die after 20 - 30 divisions because telomeres are gone * Telomerase - some cells (bone marrow, stem cells, etc...) contain this enzyme which regenerates telomeres.   * Present in 90% of cancer cells as well 

DNA Organization

* Occurs during Synthesis phase of a cell's life * This is done to prepare for cell division so

that each daughter cell is identical and contains all of the necessary chromosomes

* Occurs during 2 major stages  1. Splitting the DNA  2. Building the new strands

DNA Replication

1. Splitting the DNA * Helicase - an enzyme that breaks the hydrogen  bonds holding the two strands of DNA together.

*starts at TATA boxes

 * Replication fork - point where the DNA opens

 * Single strand binding proteins - hold the strands apart during replication

DNA ReplicationDNA replication simple http://www.dnatube.com/video/365/DNA-Replication

2. Building the new strands * Primase - enzyme that lays down a short RNA primer (necessary for DNA

polymerase to attach. This acts as a starting spot. * DNA polymerase – attaches new nucleotides to the growing strand. DNA can only add new nucleotide in the 3’ direction   

DNA Replication

* Leading and lagging strands* DNA polymerase can only add nucleotides in the 5' - 3' direction (adding to the 3’ end)

* Leading strand - runs 5' - 3'. Can be built continuously.* DNA Polymerase III – the enzyme that adds new nucleotides to build the growing LEADING strands.

 

DNA Replication

* Lagging strand - runs 3' to 5'. Must be built in chunks called Okazaki fragments

As helicase unzips the DNA strands, RNA primase lays down an RNA primer. DNA polymerase II can now attach to the 3’ end of the RNA primer and adds DNA nucleotides in the 3’ direction. The RNA primer is later replaced with DNA nucleotides by DNA polymerase I.

* Ligase - enzyme that stitches fragments of sugar-phosphate covalent bonds together

DNA Replication

DNA Replication

Extra phosphates on each nucleotide provide the energy for the process. Called "active monomers" or "phosphorylated nucleotides"

DNA replication detailed summaryhttp://www.youtube.com/watch?v=vNXFk_d6y80

DNA Replication

DNA replication real time videohttp://www.hhmi.org/biointeractive/dna-replication-advanced-detail

DNA Replication video detailed!http://www.dnatube.com/video/4197/Process-of-DNA-Replication

• DNA Replication is often referred to as

semi – conservative

 * Semi-conservative - Each new molecule of DNA is composed of one old (parent) strand one new

(daughter) strand

DNA Replication

Replication and Telomeres

• Telomere- ends of linear chromosomes with repetitive DNA sequence: TTAGGG • Telomeres allow cells to distinguish chromosome ends from broken

DNA and protect the DNA during cell division• Telomeres help to delay cell death

• Telomeres shorten with each cell division because DNA polymerase leaves 50-200 bp unreplicated on the lagging strand.• Cellular senescence is triggered when telomeres are 4-6 kb

DNA Replication and Telomeres

Telomerase- enzymes that adds telomeres to end of DNA strand.Only found in embryonic and stem cells (and cancer cells)

DNA Repair

DNA suffers from environmental stresses, radiation, UV light, and carcinogens, which modify the DNA. In addition, internal errors formed during replication must be corrected

* If any mistakes (mutations) are found, nucleases, polymerase and ligase will fix it

* Nucleases - enzymes that double check that no mistakes were made  

DNA Replication - Overview

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Molecular GeneticsDNA RNA Protein

Chapter 12

* DNA "makes us who we are" by coding for proteins

* These proteins are responsible for our various phenotypes

 * EX: DNA determines the color of the protein pigment made in your eyes and the amount

of melanin pigment made in your skin that gives you your skin color.

* Protein Synthesis - process by which a protein is made from the instructions contained in DNA

* Requires help from another nucleic acid called RNA

DNA & Proteins

* RNA = Ribonucleic Acid

* Differences between RNA and DNA 1. RNA is single stranded 2. RNA has Uracil instead of Thymine 3. RNA has ribose, not deoxyribose

* Three kinds of RNA 1. Messenger RNA (mRNA) - copies DNA's information and takes it out of the nucleus to a ribosome

 2. Transfer RNA (tRNA) - picks up and transfers Amino Acids to the ribosome  * contain an amino acid at one end and an anti-codon at the other

 3. Ribosomal RNA (rRNA) - one of the components of ribosomes helps to build the polypeptide chain/protein

RNA Structure & Function

RNA Structure & Function

* Two main stages of Protein Synthesis

 1. Transcription* Occurs in the nucleus

  * DNA  mRNA

 2. Translation* Occurs at a ribosome

  * mRNA Protein

Protein Synthesis DNA RNA PROTEIN

TRANSCRIPTION- Copying a gene from DNA to RNA

Steps *Binding/Initiation

1. Helicase splits DNA molecule  2. SSBP's hold DNA open  3. RNA Polymerase attaches to ONE of the DNA  strands at a promoter region  a. Promoter region - TATA box *Elongation

4. RNA polymerase builds a complementary mRNA strand in a 5' to 3' direction  a. Reminder: If there is an A in the DNA, a Uracil will be paired to it (no Thymine in RNA) *Termination

5. RNA stops when it reaches a termination sequence   a. sequence usually involves 'AAAAAAAA'   6. mRNA transcript detaches and DNA strands go back together 

Transcription

Transcription

mRNA must be processed before leaving the nucleus because it contains ‘junk’

* Steps  1. mRNA transcript receives a cap and tail  a) 5' cap - cap of repeated G's at 5' end   b) 3' poly- A tail - tail of repeating A's at 3' end  * These protect the molecule and control its transport out of the nucleus.

  2. Introns are excised and Exons are stitched together  a) Introns - pieces of mRNA that are not needed are  excised by

spliceosomes and stay IN the nucleus  b) Exons - needed pieces of mRNA that code for proteins. They EXIT the nucleus and are Expressed. Exons are glued together by SnRP's (small nuclear ribonucleoproteins)

* The finished product leaves the nucleus and joins w a ribosome to make proteins. The processing allows several proteins to be made from the same mRNA.

mRNA Processing

mRNA Processing

Transcription and Translation cartoon videohttps://www.youtube.com/watch?v=rKxZrChP0P4

Transcription real time videohttps://www.youtube.com/watch?v=SMtWvDbfHLo&list=PLRSu0DZ2i6tGdGB8reN_yOyfkGq_AX2mE&index=3

* Central dogma- all organisms on earth have nucleic acid (DNA/RNA) that codes for proteins.* Every three “letters” of mRNA is called a codon. Each codon codes for one specific amino acid in the protein that will eventually be made.  

DNA: TAC TGC CTC GAA GCC TCG ATC  mRNA: AUG ACG GAG CUU CGG AGC UAGProtein:

AUG Methionine (always at start of chain)

ACG ThreonineGAG Glutamic acidCUU CGG AGC UAG

Genetic Code

* Steps of Translation - (now out of the nucleus)  1. Binding/Initiation

a. mRNA & tRNA bind to small ribosomal subunit (40sv)  b. Smaller ribosomal subunit binds to larger one (60sv) 

2. Elongation a. tRNA molecules bind to mRNA based on matching their anti-codon to mRNA codon

  b. Amino acids are dropped off and hooked together via peptide bonds  c. Ribosome moves along mRNA, repeating process of bringing tRNA's in, forming peptide bond, and releasing tRNA's  

3. Termination  a. Ribosome reaches a stop codon and the ribosomal complex breaks apart

Translation

* Ribsome has three sites  1. A site: where the tRNA is accepted/attaches  2. P site: where the polypeptide (protein) is built by making a peptide bond

 3. E Site: where the tRNA exits the ribosome

Translation

Translation* A gene must start with a start codon (initiator) that tells ribosome to start translating (AUG - methionine)

* A gene will end with a stop codon (terminator) that tells the ribosome to stop translating  * There are 3 stop codons (no amino acids) (UAA, UAG, and UGA.

* Many ribosomes can translate the same mRNA transcript at the same time

Translation real time videohttps://www.youtube.com/watch?v=TfYf_rPWUdY&list=PLRSu0DZ2i6tGdGB8reN_yOyfkGq_AX2mE&index=4

Transcription and Translation cartoon videohttps://www.youtube.com/watch?v=rKxZrChP0P4

Products of Translation

* Proteins usually have to be processed before they can be used as well. This occurs at the ER

* Proteins made on free ribosomes will be used within the cell

* Proteins made on ribosomes are sent to Golgi to be shipped out of the cell or used in the cell membrane

Protein Synthesis Overview

Gene Mutations

Mutation - a change in DNA or chromosome* Causes (mutagens)   * Environmental factors  * Chemicals, sunlight  * Replication errors

* Point mutation- impacts only one base (this is the most common type of mutation)  * Substitution- a base is replaced with another

* Insertion- a new base is inserted – shifts the reading frame  * Deletion- a base is removed – shifts the reading frame

* Non-sense mutation - inserts a stop codon that prevents a protein from being made* Mis-sense mutation - changes the protein being made* Silent mutation – Change in DNA, but no change in protein due to ambiguity of code.

Mutations* Gene Mutations are usually neutral or have no affect. There are two reasons for this:    1. Mutations that occur in introns will not be expressed in the protein being made.   2. Mutations that affect the third letter in a codon often don’t change the amino acid    (Wobble effect)

* Mutations can be harmful or beneficial   * EX: make a protein work more efficiently.

* EX: make a protein inactive.

* Mutations are an important source of new genetic variety. This is where new traits come from.

DNA Mutations and Protein Synthesishttps://www.youtube.com/watch?v=GieZ3pk9YVo&index=2&list=PLIIJseG3DYUK0ijyvRoNqjMia9OvPNVjJ

 * Recessive disorders  * Sickle Cell Anemia   * point substitution of A for T on Beta-globin gene  * Cystic Fibrosis   * mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR) gene  * most common mutation is a deletion of three nucleotides that   results in a loss of the amino acid phenylalanine  * Hemophelia - MANY causes  * Tay-Sach's  * mutations of the HEXA gene on chrom. 15   * By 2000, more than 100 different kinds of mutations had been  

identified in the HEXA gene* Phenylketoneuria

  * mutation of PAH gene on chrom. 12   * More than 400 disease-causing mutations have been found in PAH gene. 

Gene Mutations

 * Dominant disorders  * Huntington's

 * All humans have the Huntingtin gene (HTT) on chrom. 4, which codes for the protein Huntingtin (Htt).

  * Part of this gene is a repeated section (CAG) called a trinucleotide repeat  * it varies in length b/w individuals and may change length between generations.   * When the length of this repeated section reaches a certain point, it produces an altered form of the protein, called mutant Huntingtin protein (mHtt)  * This leads to increase in decay rate of certain neurons  * Leads to disease symptoms  * Affects muscle coordination and leads to cognitive decline and dementia

Gene Mutations

Every cell in your body contains the same DNA, but some genes are shut off.

~For example, your eyeball cells don’t need the same proteins as your liver cells.

 ~In humans these genes can be shut off by methylating them. There are also regulatory genes that can promote or inhibit the transcription of certain genes.

Gene Regulation