DNA & Protein SynthesisChapter 18

The Molecule of Heredity

Seeking the Genetic Material• 1928 Griffith finds that virulent bacteria

can transform nonvirulent bacteria into the deadly form. Virulent: able to cause disease

• 1944 Avery: found DNA was the molecule of heredity, not protein or RNA.

• 1952 Hershey and Chase: found that viruses injected DNA into host bacteria. DNA is confirmed as the unit of heredity.

Hershey and ChaseExperiment

Animation & Game

Avery, Macleod, McCarthy

Who Else Contributed?• Chargaff: 1949 discovered there are

always equal amounts of Adenine to Thymine and the same percentage of Cytosine to Guanine.

• Rosalind Franklin 1950-53: Photographed DNA with X-rays.(pg 187, Fig 10-4) Found helictical shape (i.e. Helix Shape) while working in the lab of Maurice Wilkins.

Chargaff’s Ratios

Franklin Click:NOVA News Minute

16.7 The Structure of DNA • Structure was discovered by Watson and

Crick in 1953, 1962 receive Nobel Prize.• Double Helix: • Nucleotides: Phosphate, Sugar

(deoxyribose) and a Nitrogen base (A,T,C,G)

• DNA Backbone: Sugar-Phosphate-Base Connected by Phosphodiester linkage

• 5 carbon sugar and phosphate molecule are the same for each nucleotide. Nitrogen base changes.

Deoxyribonucleic Acid (DNA Continued)

• 2 strands one strand runs 5’ to 3’ the other 3’ to 5’ (upside down)

• Where does the 5’ and 3’ come from?Read DNA From HereSynthesize

from 5’ to 3’

Nitrogen Bases: 16.8• Adenine double bonds to Thymine

• Cytosine triple bonds with Guanine

• GCAT Bases are held together by weak hydrogen bonds.

• Purines (A&G) vs. Pyrimidines (C,T,U)

• A mistake here is a Point Mutation.

• Can you tell me the complementary strand for : AATCGCGA? ______________

Eukaryotic DNA Replication

• Overview Cinema: The Forest from the Trees

• The DNA Unzips, and each strand serves as a template for the formation of a new strand composed of complementary nucleotides G-C, A-T

HHMI Replication

Replication: Entering the Forest

• DNA Replication begins at special Sites called Origins of Replication

• Where the two strands of DNA split is called replication bubbles (thousands)

• Why do you need thousands of these bubbles? (Hint: The DNA molecule has 3 billion base pairs)

DNA Replication 16.12

• Double helix unwinds using DNA Helicase.

• DNA Helicase breaks the hydrogen bonds.

• Where the DNA breaks apart is called the replication fork. DNA polymerase (another enzyme) adds nucleotides at the 5’ to 3’ end towards the replication fork.

• FYI: Humans= 50 nuclotides/sec

DNA Replication Explained

Meselson & StahlExperiment

What about the 3’ to 5’ end?

• Need RNA Primer. (RNA and the enzyme primase).

• Lagging strand- away from the replication fork replicates using Okazaki fragments.

• Each 100-2000 nucleotide Okazaki fragment is joined by DNA ligase.

• Fig 16.15 & 16.16

Checking for errors

• DNA Polymerase also proof reads the strands- Mismatch Repair

• A mistake in nucleotide pairing is a Mutation

• Multiple replication forks happen all at once so that the process is speedy.

DNA Review

Flashcards

How do we keep the replication process clean?

• Single-stranded binding proteins scaffold the two DNA strands apart.

• Topoisomerase cuts DNA that is wound too tightly.• Telomeres, made by the enzyme telomerase

protect against gene loss at the end of the chromosome. Fig 16.19– Telomeres repeating sequences of TTAGGG (thousands

of times)– Telomeres shorten with each cell division. Aging anyone?

How do bacteria replicate their DNA? Fig. 16.2

The TechnicalDetails

The TechnicalDetails

The Non-TechnicalDetails

What is RNA and how is it useful?

• RNA= Ribonucleic Acid

• Single stranded. Ribose sugar.

• Transcribes DNA and Translates it into proteins.

• Proteins are organic compounds that have specific jobs in the cell. (Ex. Enzymes)

PBS Video

RNAi

What are the 3 types of RNA?• mRNA= Messenger RNA

– Transcribes or rewrites DNA’s message as mRNA, mRNA carries message to ribosome

• rRNA = Ribosomal RNA. Fig. 17.16– Creates the ribosomes on the rough ER and

cytoplasm where proteins are made.

• tRNA = Transfer RNA. Fig. 17.14 & 17.15– Transfers amino acids to the ribosomes and

translates the mRNA into protein. (Called Translation because the message changes from nucleic acid to protein, a different organic compound)

Transcription and TranslationCinema Fig. 17.3

• Don’t relax too much

• Pencils out? Notes ready? Lets work!

Overview Movie

Start Here

HHMi TranscriptionMore Detail

First Base

Second Base

Third Base

U C A G

  U    

UUUphenylalanine

UCUserine

UAUtyrosine

UGUcysteine

U

UUCphenylalanine

UCCserine

UACtyrosine

UGCcysteine

C

UUAleucine

UCAserine

UAAstop

UGAstop

A

UUGleucine

UCGserine

UAGstop

UGGtryptophan

G

  C    

CUUleucine

CCUproline

CAUhistidine

CGUarginine

U

CUCleucine

CCCproline

CAChistidine

CGCarginine

C

CUAleucine

CCAproline

CAAglutamine

CGAarginine

A

CUGleucine

CCGproline

CAGglutamine

CGGarginine

G

  A    

AUUisoleucine

ACUthreonine

AAUasparagine

AGUserine

U

AUCisoleucine

ACCthreonine

AACasparagine

AGCserine

C

AUAisoleucine

ACAthreonine

AAAlysine

AGAarginine

A

AUG (start)methionine

ACGthreonine

AAGlysine

AGGarginine

G

  G    

GUUvaline

GCUalanine

GAUaspartate

GGUglycine

U

GUCvaline

GCCalanine

GACaspartate

GGCglycine

C

GUAvaline

GCAalanine

GAAglutamate

GGAglycine

A

GUGvaline

GCGalanine

GAGglutamate

GGGglycine

G

AUG (start)methionine

UAA UGA UAG (stop)

What is Wobble?

• A relaxation of the base pairing rule. – Example: UCU, UCC, UCA, UCG = serine

• Why is their Wobble room?– Allows for point mutations not to cause an

incorrect protein to be made.– Since there are only 20 Amino Acids and 64

codons to code for them we can have repeats.

What are the three stages of transcription?

• Initiation: RNA polymerase binds to the DNA promoter. Fig 17.8– TATA box is the area that transcription factors

recognize within the promoter. – Creates a transcription initiation complex

(transcription factors & RNA polymerase bond on promoter)

• Elongation: DNA template continues to grow as RNA polymerase separates DNA strand. – Transcribed part: Transcription unit (made of codons)

(1 gene transcribed many times in caravan fashion)

• Termination: mRNA is cut free from DNA template

How do Eukaryotes control Gene expression?

• Transcription creates Pre-mRNA. Fig 17.10

• Pre-mRNA includes Introns and Exons• Introns= Fillers (intervening sequences)

• Exons= Code for proteins (expressed sequences)

• mRNA is just the Exons

Eukaryotic Transcription

How else is mRNA processed for translation?

• snRNP’s (“snurps”) & splicesomes remove introns, creating the final mRNA sequence= Exons only. Fig. 17.11

• What stops the mRNA from degrading by hydrolytic enzymes & binds it to the ribosome?– 5’ cap: modified guanine nucleotide is added to

the 5’ end. – A poly tail: 30-200 adenine nucleotides on the

3’ end. (Also helps release mRNA into the cytoplasm.

What do enhancers do? Fig 18.9

• Enhancer’s causes the gene it is enhancing to be expressed. Transcription occurs in eukaryotes when an enhancer site is activated.

Fig. 18-9-3

Enhancer TATAbox

PromoterActivators

DNAGene

Distal controlelement

Group ofmediator proteins

DNA-bendingprotein

Generaltranscriptionfactors

RNApolymerase II

RNApolymerase II

Transcriptioninitiation complex RNA synthesis

HHMI TranscriptionBasic

Fig. 18-8-3

Enhancer(distal control elements)

Proximalcontrol elements

Poly-A signalsequence

Terminationregion

DownstreamPromoter

UpstreamDNA

ExonExon ExonIntron Intron

Exon Exon ExonIntronIntron Cleaved 3 endof primarytranscript

Primary RNAtranscript

Poly-Asignal

Transcription

5

RNA processing

Intron RNA

Coding segment

mRNA

5 Cap 5 UTRStart

codonStop

codon 3 UTR Poly-Atail

3

Putting it all together

And now Translation Fig. 17.17-17.19• Translation: mRNA codons code for an Amino

acid sequence.• tRNA attaches to the ribosomal rRNA with

anticodon. GTP recycles tRNa with enzyme.• Ribosome: protein and rRNA created in the

nucleolus.– Bacterial & Eukaryotic ribosomes differ, this allows

Tetracycline and Streptomycin to stop protein production in bacterial ribosomes.

– Small and large subunits for attachment to mRNA. – Three binding sites for tRNA

• P site: Holds the growing polypeptide chain• A site: Holds the newest tRNA• E site: Exit site

What are the three stages of translation?

• Initiation: mRNA’s AUG codon (methionine) attaches to the ribosome. tRNA attaches.

• Elongation: forms the polypeptide chain as tRNA’s continue to attach.

• Termination: mRNA reaches a stop codon. Release factor hydrolyzes the bond. mRNA is degraded. Polypeptide is freed from ribosome. – After this the polypeptide will fold or pleat (secondary

structure), Chaperonine will complete the tertiary structure.

How Proteins fold Fold it, the Game!!!

What about when a mistake occurs?

• They can effect the species gene pool.

• Point mutation= single base-pair substitution. (sickle cell)

• Insertion or deletion= frameshift mutations. The Dog -> HeD og

• Missence Mutation= If a codon within a gene turns into a stop codon translation is altered.

Gel Electrophoresis

• How does this link to Gel Electrophoresis?

• Below is a reminder of what Electrophoresis is and how it works.

Gel Electrophoresis