Molecular GeneticsNucleic Acids

http://learn.genetics.utah.edu/ Learn Genetics University of Utah Genetics Learning Center Ch 12,13,15

The Central Dogma of Molecular Biology

DNA

RNA

Protein Synthesis

1957…Crick

Milestones in DNA History Test Text- p263- Table 12-1

• 1869/1871 Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA.

(Nuclein = DNA, RNA, Proteins)

• *what they knew: Pasteur 1862…germ theory, No ‘spontaneous generation’ Mendel ‘rediscovered’ 1900’s ……. Fleming 1928…penicillin Vaccines/Meds…

• *genes stored genetic information…Proteins/20 different amino acids, ’DNA’/simple base unit….so….proteins must store the genes.

Milestones in DNA History• 1928 Franklin Griffith, a British medical officer, discovers that

genetic information can be transferred from heat-killed bacteria cells to live ones. This phenomenon, called transformation, provides the first evidence that the genetic material is a heat-stable chemical. (Text-p261)

*Griffith was trying to develop a vaccine against pneumonia when he serendipitously discovered the phenomenon of transformation.

….scientists hypothesized that a chemical substance (a transforming principle) was transferred from the dead bacteria to the living cells and caused transformation

Griffiths experiments- 1928• Textp261Although neither the rough strain nor the heat-killed strain smooth strain could kill a mouse, a combo of the two did. Autopsy showed the presence of living S-strain cells in the dead mice…results indicated that some substance in the heat-killed S cells had transformed the living R cells into a virulent form.

“transforming principle”

Milestones in DNA History• 1944 Oswald Avery, Maclyn McCarty and Colin MacLeod, identify

Griffith's transforming agent as DNA. Their discovery is greeted with skepticism, in part because many scientists still believe that DNA is too simple a molecule to be the genetic material.

By lysing S cells, separatingthe contents- lipids, proteins,polysaccharides, nucleic acids(DNA,RNA) and testing eachfraction to see if it couldtransform living R cells intoS cells. Only when R cells were treated with the nucleicacids from the S cells werethey transformed.…still…it’s the protein

Milestones in DNA History• 1952: Alfred Hershey and Martha

Chase (Text-p 262)

• Demonstrated that DNA, not protein, is involved in viral reproduction.

The “transforming agent”• Tagged the protein coat of one

bacteriophage (virus that infects bacteria) with one radioactive isotope, and the viral DNA of a second sample with a different isotope (red)

• Infected, Blended, centrifuged: protein still outside the cell, DNA in the portion with the bacterial cells

Race is on…....Discovery of the structure of DNA?

Milestones in DNA HistoryThe Necessary Groundwork….• 1912 Physicist Sir William Henry Bragg, and his son, Sir William

Lawrence Bragg, discover that they can deduce the atomic structure of crystals from their X-ray diffraction patterns. This scientific tool will be key in helping Watson and Crick determine DNA's structure.

• “Crystallography”(Text p 265)

Milestones in DNA HistoryThe Necessary Groundwork….• 1952 Maurice Wilkins and Rosalind

Franklin/ Franklin and Raymond Gosling: work on X-ray diffraction experiments on DNA. Thanks largely to Franklin’s expertise in crystallography - the vital “photo 51” was taken in May 1952.

• Watson and Crick cracked the structure of DNA in March 1953. The spur for Jim Watson's new attempt at model building had been seeing the clear helical pattern of "photo 51" and deciding to discard experimental data pointing to three chains and opt for two.

Milestones in DNA HistoryThe Necessary Groundwork….• 1949 Erwin Chargaff: Biochemist. Reports that the composition of

DNA is specific; amount of adenine equals the amount of thymine and the amount of guanine equals the amount of cytosine in DNA from EVERY species………..Helix becomes a double helix

• 1953 James Watson and Francis Crick discover the molecular structure of DNA.

Compiled data/made model“The forest through the trees”

Milestones in DNA History

• 1962 Francis Crick, James Watson, and Maurice Wilkins receive the Nobel Prize for determining the molecular structure of DNA.

http://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html~14 minutes"This structure has novel features which are of considerable biological interest"

Great Discoveries in Science: The Double Helix 16.53

DNA vs RNA• Double Helix vs Single strand• DNA- deoxyribose sugar (one less O than ribose) RNA- Ribose sugar• 4 Nitrogen Bases

DNA: A-T C-G

RNA: A-UC-G

• DNA stays in the nucleus,RNA made in the nucleus, works in the cytoplasm… where?• DNA contains information to

direct development; RNA: 3 main types carry out DNA directions:

mRNA = messengertRNA = transferrRNA = ribosomal

Structure of DNA• DNA is made up of nucleotides, each consisting of:

• A sugar “deoxyribose”• A phosphate group• A nitrogenous base:

• Adenine=Thymine• Cytosine =Guanine

• Covalently Bonded: “the brick”

• # Carbons?

O

1

23

5

4

DNA at the molecular level

C5’ 5’ prime end (Phosphate Group)

C1’(Nitrogenous base)

C3’ (OH Group) 3’ end

Hydrogen Bonds

Anti-parallel Orientation of DNA• 5’ end:

Phosphate group• 3’ end: OH group• Antiparallel• Hydrogen

Bonding between- individually weak, collectively strong

• Built in a 5’ to 3’ direction,Read 3’ to 5’

BUILD READ

Note: Difference in shape between A, G and T,C?

Purines- 2 rings, Pyrimidines- 1ringPurine bonds with a Pyrimidine (3ring)

Structure of DNA• http://www.hhmi.org/biointeractive/disease/dna_chem_struc

ture.html(play Large WMV)

2.44

Semiconservative Replication

• One strand of parent in EACH new strand• Explains the perpetuation of mutationsTEXT p268

Meselson-Stahl Experiment 1958 Disproves Conservative (b), dispersive (c)

DNA Replication

Replication Fork-Happening at many places along the molecule

VIDEO: REPLICATION ENZYMES

DNA Replication FactoryReplication in action 7,8

DNA Replication: Replicating enzymes- Unwinding of DNA1. Initiator Protein: recognizes where to begin2. Helicase Enzyme: Lands at the origin- starts

breaking the Hydrogen bonds between the bases, *working in both directions, at the replication fork

3. SSBP’s (Single Stranded Binding Proteins)-keep the strands from joining again

4. Primase Enzyme: the RNA “primer”/marker on the open end- marks where to begin adding nucleotides

5. DNA Polymerase: adds complimentary nucleotides, one at a time (A to T, G to C)

DNA Replication: Replicating the strands: Leading vs Lagging

Replication Bubble

VIDEO: REPLICATING THE STRANDS

DNA Replication: Replicating the strands: Leading vs Lagging

LEADING STRAND: Builds towards the replication fork in a continuous manner, reading 3’ to 5’ and building 5’ to 3’

LAGGING STRAND: Builds away from the replication fork in a discontinuous manner creating, using DNA primase at each starting point.

OKAZAKI FRAGMENTS –segments of DNADNA LIGASE- welds together the okazaki fragments into a continuous strand.

DNA Replication: The Twisting Problem

TOPOISOMERASE: Enzyme that prevents kinking of the DNA strand as it unwinds

VIDEO: THE TWISTING PROBLEM

DNA Replication

Proofreading and Repair

“Enzymes on Patrol”• Reduces the rate of

error to ~ 1/billion base pairs• Mutations:

harmful, fatal or just part of the evolutionary process

VIDEO: PROOFREADING and REPAIR

Backs up, replaces with correct base

RNA SYNTHESIS• Messenger RNA (mRNA)• Single, uncoiled chain• Carries genetic information from the nucleus to the

ribosomes

• Transfer RNA (tRNA)• Single chain folded into a hairpin shape• Carries amino acids from the cytoplasm to the

ribosomes

• Ribosomal RNA (rRNA)• Globular• Found in the ribosome and assists in protein synthesis

HANDOUT: How DNA makes RNA

THEMAKING

OF A

PROTEIN

1. Transcription- mRNA

• Transcription (animation-next concept) http://www.phschool.com/science/biology_place/biocoach/transcription/tcproc.html

Video: Main Menu>Genetic Code & Protein Synthesis>RNA>Transcription

Process of copying DNA into mRNA:Every 3 bases will code for 1 amino acid…..building a protein

2. Translation- tRNA*DNA base sequence (triplets) of the “gene” coding for the synthesis of a particular polypeptide chain.

*Base sequence (codons) of the transcribed mRNA

*Consecutive base sequences of tRNA anticodons capable of recognizing the mRNA codons “calling” for the amino acids they transport *Translation BEGINS when mRNA LEAVES the nucleus

2. Translation- tRNA• Begins once mRNA leaves the nucleus.• Ribosomes in the cytosol make proteins for the cell; • Ribosomes on the rough ER make proteins for export.

The Genetic Code: Specific for Amino AcidsDNA: Triplets TACmRNA: Codons AUGtRNA: Anticodons UAC= Amino Acid: Methionine (start)

**The CODON determines the Amino Acid NOT the anticodon

• # of different Amino Acids?• Possible Code Combinations?

Ribosomal Subunits, mRNA and tRNA

• Large Ribosomal subunit

• Small Ribosomal subunit

• P site• A Site• tRNA: AA

attachment site; anticodon

Translation1. Codon recognition:

translation begins2. Peptide Bond formed

between the amino acids

3. Translocation: Ribosome moves AHEAD; what was on the P site is now on the growing peptide chain, the tRNA on the A site moves to the Psite, making room for the next AA

4. Completing the protein: Stop codon- protein is released

Video: Main Menu>The Genetic Code & Protein Synthesis>Translation, Protein Synthesis

Codons:To find:ACG

“First position”-letter gives you the correct row.

“Second position” –letter gives you the correct column

“Third position” –final row

Codon for Threonine

•Ribosome shifts one codon in the 3’ direction•Stop codon: no anticodon

TRANSLATIONhttp://bcove.me/lz405t1m 2.06

Summary

Worksheet

The mRNA Editing Process

Video: Main Menu>The Genetic Code & Protein Synthesis>Exons and Introns

• A strand of mRNA inside the nucleus is much longer than the segment that actually leaves. It is made up of two parts:

• Exons: Proteins Coding Region of a Eukaryotic Gene. Exons are the part that exits the nucleus.

• Introns: Non-protein Coding Regions of a Eukaryotic Gene- Introns are the part that remain inside the nucleus. Usually longer. Thought to be “junk DNA”, now know this “silent DNA” is important in gene expression

Transcribed mRNA in the nucleus

Exons and Introns

*Average transcrip

t

27,000 nucleotides.

Average protein

1,200 nucleotides

The mRNA Editing Process • Once the mRNA strand is ready to leave the nucleus (only Exons), a

cap and a tail are placed on the ends of the mRNA to prevent the genes from being damaged

• All mRNA are read in a 5’ to 3’ direction

• G Cap: (Guanine)- placed at the 5’ end of the RNA

• Poly A Tail: (Adenine)- placed at the 3’ end of the RNA

Mutations

*Substitutions*Insertions*Deletions

• Random mistake (“spontaneous”) made in the DNA copying process; not planned, not predictable

• Even with the “Enzymes on Patrol” errors can still occur• Causes:?...... • UV, drugs, alcohol, X-Ray, chemicals: (“Mutagens”), viruses• Some are very harmful…where the mutation occurs on the DNA chain

can change its effect. Depends on when occurs in formation of the zygote, through the adult organism

• Basic Types: Base/Point and Frameshift Mutations

Video: Main Menu>The Genetic Code & Protein Synthesis>Mutations

• Silent mutations code for the same amino acid. • Nonsense mutations code for a stop codon, which may cause a shortened protein. Depending on

the extent of protein loss, protein function may be affected. • Missense mutations:

• Conservative mutations cause a change in amino acid but no change in properties, as compared to the wild-type.

• Non-conservative mutations induce different properties compared to the wild type. T The gly → valine transition resulting in a protooncogene → oncogene transition is an example of this type.

2. Point Mutations• Substitute one nucleotide for another• Usually not very harmful• Ex: THE CAT ATE THE RAT

THE CAT ATE THE NAT

3. Frameshift Mutation • One or two nucleotide pairs are inserted or

deleted from the molecule, altering the reading frame• Very harmful

Ex: THE CAT ATE THE RAT ……….CA Deleted

THE TAT ETH ENA T

Gene Regulation: Operons Text p 307

• Two Examples of Regulatory Systems:1. Inducible- usually in the ‘off’ position2. Repressible- usually in the ‘on’ position …Looking at one example of each

•Operons: functionally related genes regulated together in gene complexes. 1960’s Ecoli Bacteria

The lac operon “inducible system”

• Found in prokaryotic cells• Regulates synthesis of inducible enzymes• Usually codes for enzymes in a catabolic pathways• Usually in the ‘off’ position

Controlled by a repressor keeping it in the ‘off position

• LACTOSE PRESENT: Repressor is inactive; the operon is ON.• Enzymes made to break down lactose• Helps cell save the energy costs of making enzymes when

no substrates (like lactose) are available

The lac operon “inducible system”

Video: Main Menu>The Genetic Code & Protein Synthesis>Gene Regulation in Prokaryotes

*Noncompetetive site on the enzyme?*

Trp operon “repressible system” p310”

*Tryptophan- an amino acid bacteria need most of the time*Anabolic pathways*Usually ON