Chapter 17: Chapter 17: From Gene to ProteinFrom Gene to Protein(Protein Synthesis)(Protein Synthesis)

Essential Knowledge

3.a.1 – DNA, and in some cases RNA, is the primary source of heritable information (17.1-17.4).

3.c.1 – Changes in genotype can result in changes in phenotype (17.5).

Question? Question? How does DNA control a cell? (or identify a

phenotype) By controlling protein synthesis (otherwise

known as gene expression) Proteins are the link between genotypegenotype and

phenotypephenotype

1909 - Archibald 1909 - Archibald GarrodGarrod Suggested genes control enzymes that

catalyze chemical processes in cells Inherited Diseases - “inborn errors of

metabolism” where a person can’t make an enzyme Symptoms reflect person’s inability to make proteins/enzymes

ExampleExample Alkaptonuria - where urine turns black

after exposure to air Lacks - an enzyme to metabolize/break

down alkapton

George Beadle and Edward George Beadle and Edward TatumTatum

Worked with Neurospora and proved the link between genes and enzymes

Grew Neurospora on agar Varied the nutrients in the agar Looked for mutants that failed to grow

on minimum agar

ConclusionConclusion

Mutations were abnormal genes Each gene dictated the synthesis/production

of one enzyme One Gene - One Enzyme Hypothesis

Current HypothesisCurrent Hypothesis One Gene - One Polypeptide

Hypothesis.Why change? Not all proteins are enzymes

We now know proteins may have 4th degree structure.

Central DogmaCentral DogmaDNA

Transcription

RNA Translation

Polypeptide chain (will become protein)

ExplanationExplanation DNADNA – the genetic code or genotype RNARNA - the message or instructions PolypeptidePolypeptide - the end product for the

phenotype

Why is there an RNA Why is there an RNA intermediate?intermediate? Evolutionary adaptation:

Check-point in processProvides protection for DNA codeMore copies can be made simultaneously

Genetic CodeGenetic Code

Sequence of DNA bases that describe which amino acid to place in what order in a polypeptide chain

The genetic code gives ONLY the primary protein structure All other protein structures result from chemical interactions

amongst primary protein structure

Genetic CodeGenetic Code

Is based on triplets of bases (called codonscodons)

Has redundancy; some AA's have more than 1 code/3-base codon

ProofProof - make artificial RNA and see what AAs are used in protein synthesis (early 1960’s)

Codon Codon A 3-nucleotide “word” in the Genetic

Code 64 possible codons known

Codon

Amino

acid

Codon DictionaryCodon Dictionary Start- AUG (Met) Stop- UAA

UAG UGA

60 codons for the other 19 AAs

Code RedundancyCode Redundancy

Third base in a codon shows "wobble” effect

First two bases are the most important in reading the code and giving the correct AA The third base often doesn’t matterThis allows for mistakes during DNA

replication

Reading FrameReading Frame The “reading” of the code is every three

basesEx: the red cat ate the ratEx: ATT GAT TAC ATT

The “words” (codons) only make sense if “read” in this grouping of three (in correct “letter” order)

Code EvolutionCode Evolution

The genetic code is nearly universal Ex: CCG = proline (all life) Reason:

Code must have evolved early Life on earth must share a common ancestor

Protein Synthesis IntroProtein Synthesis Intro Intro movie

Protein Synthesis Protein Synthesis IntroIntro Step 1: Transcription:

DNA mRNA Step 2: Translation:

mRNA tRNA Am. Acid Polypep. chain

Polypeptide chain then becomes protein

TranscriptionTranscription Process of making RNA from a DNA

templateRNA type: mRNA (messenger)Intermediate type

Takes place in nucleus (in eukaryotes)

Transcription StepsTranscription Steps1. RNA Polymerase Binding

2. Initiation

3. Elongation

4. Termination

RNA PolymeraseRNA Polymerase

Enzyme for building RNA from RNA nucleotides Prokaryotes - 1 type Eukaroyotes- 3 types

Splits two DNA strands apart Hooks RNA nucleotides together (as they

pair with DNA)

11stst Step Step: RNA Polymerase : RNA Polymerase BindingBinding

Requires that the enzyme find the “proper” place on the DNA to attach and start transcription

Different from DNA polymerase Doesn’t require an RNA primer

RNA Polymerase Binding RNA Polymerase Binding Needs:Needs:

Promoter RegionsPromoter Regions (on the DNA)Special sequences of DNA nucleotides that

“tell” cell where transcriptiontranscription begins

Transcription FactorsTranscription FactorsProteins

PromotersPromoters

Regions of DNA where RNA Polymerases can bind

About 100 nucleotides long. Include initiation site and recognition areas for RNA Polymerase

Also “decide” which DNA strand to use

TATA BoxTATA Box

ONLY in eukaryotes Short segment of T,A,T,A Located 25 nucleotides upstream from the

initiation site Recognition site for transcription factors to bind

to the DNA

Transcription FactorsTranscription Factors Proteins that bind to DNA before RNA

Polymerase Recognizes TATA box, attaches, and

“flags” the spot for RNA PolymeraseRNA poly won’t attach unless these are

present

Transcription Initiation Transcription Initiation ComplexComplex

The complete assembly of 1) transcription factors and 2) RNA Polymerase

Bound to the promoter area of the DNA to be transcribed

22ndnd Step Step: Initiation: Initiation 2nd step of transcription Actual unwinding of DNA to start RNA

synthesis. Requires Initiation Factors

CommentComment Getting Transcription started is

complicated Gives many ways to control which

genes are decoded and which proteins are synthesized

33rdrd Step Step: Elongation: Elongation 3rd step in transcription RNA Polymerase untwists DNA 1 turn at a time Exposes 10 DNA bases for pairing with RNA

nucleotides

ElongationElongation Adds nucleotides to 3` end of growing

RNA strand Enzyme moves 5` 3` (of RNA strand) Rate is about 60 nucleotides per second

CommentComment Each gene can be read by sequential

RNA Polymerases giving several copies of RNA

Result - several copies of the protein can be made

44thth Step Step: Termination: Termination DNA sequence that tells RNA Polymerase

to stop Ex: AATAAA RNA Polymerase detaches from DNA

after closing the helix

Final ProductFinal Product Pre-mRNA This is a “raw” RNA that will need

processing and modifications

Modifications of RNAModifications of RNA1. 5’ Cap

2. Poly-A Tail

3. Splicing

5' Cap5' Cap Modified Guanine nucleotide added to

the 5' end Protects mRNA from digestive enzymes Recognition sign for ribosome

attachment

Poly-A TailPoly-A Tail 150-200 Adenine nucleotides added to

the 3' tail Protects mRNA from digestive enzymes. Aids in mRNA transport from nucleus.

RNARNA SplicingSplicing Removal of non-protein coding regions

of RNA Coding regions are then spliced back

together

Introns and ExonsIntrons and Exons Introns:

Intervening sequencesRemoved from RNA.

Exons:Expressed sequences of RNATranslated into AAs

Introns - FunctionIntrons - Function Left-over DNA (?) Way to lengthen genetic message Old virus inserts (?) Way to create new proteins

Translation Poster Requirements 1. What is translation? (definition) 2. What is needed? 3. Specifics & Structure of tRNA 4. Where does it occur? 5. Ribosome specifics – be sure to include the

specifics of each subunit 6. Steps of translation & details of each step 7. What bonds are formed? 8. Illustration

22ndnd step of Protein step of Protein Synthesis: Synthesis: TranslationTranslation Process by which a cell interprets a

genetic message and builds a polypeptide

Location: mRNA moves from nucleus to cytoplasm and ribosomes

Materials Required for Materials Required for translationtranslation tRNA Ribosomes mRNA

Transfer RNA = tRNATransfer RNA = tRNA Made by transcription About 80 nucleotides long Carries AA for polypeptide synthesis

Structure of tRNAStructure of tRNA Has double stranded regions and 3

loops. AA attachment site at the 3' end. 1 loop serves as the AnticodonAnticodon.

AnticodonAnticodon Region of tRNA that base pairs to mRNA codon Usually is a compliment to the mRNA bases, so

reads the same as the DNA codon Example:

DNA- GAC mRNA – CUGtRNA anticodon - GAC

RibosomesRibosomes Two subunits (large and small) made in

the nucleolus Made of rRNA (60%)and protein (40%) rRNA is the most abundant type of RNA

in a cell

Large SubunitLarge Subunit Has 3 sites for tRNA. P site: PPeptidyl-tRNA site - carries the

growing polypeptide chain A site: AAminoacyl-tRNA site -holds the tRNA

carrying the next AA to be added E site: EExit site

Translation StepsTranslation Steps1. Initiation

2. Elongation

3. Termination

InitiationInitiation Brings together:

mRNAA tRNA carrying the 1st AA2 subunits of the ribosome

Initiation Steps:Initiation Steps:1. Small subunit binds to the

mRNA

2. Initiator tRNA (Met, AUG) binds to mRNA

3. Large subunit binds to mRNA

Initiator tRNA is in the P-site

InitiationInitiation Requires other proteins called "Initiation

Factors” GTP used as energy source

Elongation Steps:Elongation Steps:1. Codon Recognition

2. Peptide Bond Formation

3. Translocation

Codon RecognitionCodon Recognition tRNA anticodon matched to mRNA

codon in the A site

Peptide Bond Peptide Bond FormationFormation

A peptide bond is formed between the new AA and the polypeptide chain in the P-site

Bond formation is by rRNA acting as a ribozyme

After bond formation:The polypeptide is now transferred from the tRNA

in the P-site to the tRNA in the A-site

TranslocationTranslocation tRNA in P-site is released Ribosome advances 1 codon, 5’ 3’ tRNA in A-site is now in the P-site Process repeats with the next codon

TerminationTermination Triggered by stop codons Release factor binds in the A-site

instead of a tRNA H2O is added instead of AA, freeing the

polypeptide Ribosome separates

PolyribosomesPolyribosomes Cluster of ribosomes all reading the

same mRNA Another way to make multiple copies of

a protein

Prokaryotes: Prok. vs. Euk. Prokaryotes: Prok. vs. Euk. Protein Synthesis VideoProtein Synthesis Video

Polypeptide vs. ProteinPolypeptide vs. Protein Polypeptide usually needs to be

modified before it becomes functional Ex:

Sugars, lipids, phosphate groups addedSome AAs removedProtein may be cleavedJoin polypeptides together (Quaternary

Structure)

MutationsMutations Changes in the genetic make-up of a

cell Chapter 15 covered large-scale

chromosomal mutations (Hint - review these!)

Mutation types - Mutation types - CellsCells Somatic cells or body cells – not

inherited Germ Cells or gametes - inherited

Point or Spot Point or Spot MutationsMutations Changes in one or a few nucleotides in the genetic code

Effects - none to fatal

Types of Point Types of Point MutationsMutations

1. Base-Pair Substitutions

2. Insertions

3. Deletions

Base-Pair SubstitutionBase-Pair Substitution The replacement of 1 pair of nucleotides

by another pair Ex: Sickle cell anemia

Types of SubstitutionsTypes of Substitutions1. MissenseMissense - altered codons, still code for

AAs but not the right ones

2. NonsenseNonsense - changed codon becomes a stop codon

Question?Question? What will the "Wobble" Effect have on

Missense? If the 3rd base is changed, the AA may

still be the same and the mutation is “silent”

Missense EffectMissense Effect Can be none to fatal depending on

where the AA was in the protein Ex:

If in an active site - major effectIf in another part of the enzyme - no effect

Nonsense EffectNonsense Effect Stops protein synthesis Leads to nonfunctional proteins unless

the mutation was near the very end of the polypeptide

Sense MutationsSense Mutations The changing of a stop codon to a

reading codon Result - longer polypeptides which may

not be functional Ex. “heavy” hemoglobin

Insertions & DeletionsInsertions & Deletions The addition or loss of a base in the

DNA Cause frame shifts and extensive

missense, nonsense or sense mutations

Frame ShiftFrame Shift The “reading” of the code is every three

bases Ex: the red cat ate the rat Ex: thr edc ata tat her at The “words” only make sense if “read” in this

grouping of three

Question?Question? Loss of 3 nucleotides is often not a

problem Why?

Because the loss of a 3 bases or one codon restores the reading frame

MutagensMutagens Materials that cause DNA changes

1. Radiationex: UV light, X-rays

2. Chemicalsex: 5-bromouracil

Chernobyl video

SummarySummary Recognize the relationship between genes

and enzymes (proteins) as demonstrated by the experiments of Beadle and Tatum.

Identify the flow of genetic information from DNA to RNA to polypeptide (the “Central Dogma”).

Read DNA or RNA messages using the genetic code.

Recognize the steps and procedures in transcription.

SummarySummary Identify methods of RNA modification. Recognize the steps and procedures

in translation. Recognize categories and

consequences of base-pair mutations. Identify causes of mutations. Be able to recognize and discuss

“What is a gene?”