Chapter 15 From Genes to Proteins. Question? u How does DNA control a cell? u By controlling Protein Synthesis. u Proteins are the link between genotype.

Chapter 15From Genes to

Proteins

Question?

How does DNA control a cell? By controlling Protein

Synthesis. Proteins are the link between

genotype and phenotype.

For tests:

Name(s) of experimenters Outline of the experiment Result of the experiment and

its importance

1909 - Archibald Garrod

Suggested genes control enzymes that catalyze chemical processes in cells.

Inherited Diseases - “inborn errors of metabolism” where a person can’t make an enzyme.

Example

Alkaptonuria - where urine turns black after exposure to air.

Lacks - an enzyme to metabolize alkapton.

George Beadle and Edward Tatum

Worked with Neurospora and proved the link between genes and enzymes.

Neurospora

Pink bread mold

Experiment

Grew Neurospora on agar. Varied the nutrients. Looked for mutants that

failed to grow on minimum agar.

Results

Three classes of mutants for Arginine Synthesis.

Each mutant had a different block in the Arginine Synthesis pathway.

Conclusion

Mutations were abnormal genes.

Each gene dictated the synthesis of one enzyme.

One Gene - One Enzyme Hypothesis.

Current Hypothesis

One Gene - One Polypeptide Hypothesis (because of 4th degree structure).

Central DogmaDNA

Transcription

RNA Translation

Polypeptide

Explanation

DNA - the Genetic code or genotype.

RNA - the message or instructions.

Polypeptide - the product for the phenotype.

Genetic Code

Sequence of DNA bases that describe which Amino Acid to place in what order in a polypeptide.

The genetic code gives the primary protein structure.

Code Basis

If you use: 1 base = 1 amino acid 4 bases = 4 amino acids 41 = 4 combinations, which

are not enough for 20 AAs.

If you use:

2 bases = 1 amino acid 42 = 16 amino acids Still not enough combinations.

If you use:

3 bases = 1AA 43 = 64 combinations More than enough for 20

amino acids.

Genetic Code

Is based on triplets of bases. Has redundancy; some AA's

have more than 1 code. Proof - make artificial RNA and

see what AAs are used in protein synthesis (early 1960’s).

Codon

A 3-nucleotide “word” in the Genetic Code.

64 possible codons known.

Codon Dictionary

Start- AUG (Met) Stop- UAA

UAG UGA

60 codons for the other 19 AAs.

For Testing:

Be able to “read” a DNA or RNA message and give the AA sequence.

RNA Genetic Code Table will be provided.

Code Redundancy

Third base in a codon shows "wobble”.

First two bases are the most important in reading the code and giving the correct AA. The third base often doesn’t matter.

Code Evolution

The genetic code is nearly universal.

Ex: CCG = proline (all life) Reason - The code must have

evolved very early. Life on earth must share a common ancestor.

Reading Frame and Frame Shift

The “reading” of the code is every three bases (Reading Frame)

Ex: the red cat ate the rat Frame shift – improper groupings

of the bases Ex: thr edc ata tat her at The “words” only make sense if

“read” in this grouping of three.

Transcription

Process of making RNA from a DNA template.

Transcription Steps

1. RNA Polymerase Binding

2. Initiation

3. Elongation

4. Termination

RNA Polymerase

Enzyme for building RNA from RNA nucleotides.

Binding

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

Binding

Is a complicated process Uses Promoter Regions on

the DNA (upstream from the information for the protein)

Requires proteins called Transcription Factors.

Transcription Initiation Complex

The complete assembly of transcription factors and RNA Polymerase bound to the promoter area of the DNA to be transcribed.

Initiation

Actual unwinding of DNA to start RNA synthesis.

Requires Initiation Factors.

Elongation

RNA Polymerase untwists DNA 1 turn at a time.

Exposes 10 DNA bases for pairing with RNA nucleotides.

Elongation

Enzyme moves 5’ 3’. Rate is about 60 nucleotides

per second.

Comment

Each gene can be read by sequential RNA Polymerases giving several copies of RNA.

Result - several copies of the protein can be made.

Termination

DNA sequence that tells RNA Polymerase to stop.

Ex: AATAAA RNA Polymerase detaches

from DNA after closing the helix.

Final Product

Pre-mRNA This is a “raw” RNA that will

need processing.

Modifications of RNA

1. 5’ Cap

2. Poly-A Tail

3. Splicing

5' Cap

Modified Guanine nucleotide added to the 5' end.

Protects mRNA from digestive enzymes.

Recognition sign for ribosome attachment.

Poly-A Tail

150-200 Adenine nucleotides added to the 3' tail

Protects mRNA from digestive enzymes.

Aids in mRNA transport from nucleus.

Let’s see Transcription in motion…

http://www.hhmi.org/biointeractive/media/DNAi_transcription_vo2-lg.mov

RNA Splicing

Removal of non-protein coding regions of RNA.

Coding regions are then spliced back together.

Introns

Intervening sequences. Removed from RNA.

Exons

Expressed sequences of RNA.

Translated into AAs.

Spliceosome

Cut out Introns and join Exons together.

Made of snRNA and snRNP.

Result

Ribozymes

RNA molecules that act as enzymes.

Are sometimes Intron RNA and cause splicing without a spliceosome.

Introns - Function

Left-over DNA (?) Way to lengthen genetic

message. Old virus inserts (?) Way to create new proteins.

Final RNA Transcript

Alternative Splicing

The RNA can be spliced into different mRNA’s.

Each different mRNA produces a different polypeptide.

Ex. – variable regions of antibodies.

Another Example

Bcl-XL – inhibits apoptosis

Bcl-XS – induces apoptosis

Two different and opposite effects!!

DSCAM Gene

Found in fruit flies Has 100 potential splicing sites. Could produce 38,000 different

polypeptides Many of these polypeptides have

been found

Commentary

Alternative Splicing is going to be a BIG topic in Biology.

About 60% of genes are estimated to have alternative splicing sites.

One gene does not equal one polypeptide.

Translation

Process by which a cell interprets a genetic message and builds a polypeptide.

Materials Required

tRNA Ribosomes mRNA

Transfer RNA = tRNA

Made by transcription. About 80 nucleotides long. Carries AA for polypeptide

synthesis.

Structure of tRNA

Has double stranded regions and 3 loops.

AA attachment site at the 3' end.

1 loop serves as the Anticodon.

Anticodon

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 - CUG tRNA anticodon - GAC

Comment

"Wobble" effect allows for 45 types of tRNA instead of 61.

Reason - in the third position, U can pair with A or G.

Inosine (I), a modified base in the third position can pair with U, C, or A.

Importance

Allows for fewer types of tRNA.

Allows some mistakes to code for the same AA which gives exactly the same polypeptide.

Aminoacyl-tRNA Synthetases

Family of Enzymes. Add AAs to tRNAs. Active site fits 1AA and 1 type of

tRNA. Uses a “secondary genetic” code

to load the correct AA to each tRNA.

Ribosomes

Two subunits made in the nucleolus.

Made of rRNA (60%)and protein (40%).

rRNA is the most abundant type of RNA in a cell.

Large subunit

Proteins

rRNA

Both sununits

Large Subunit Has 3 sites for tRNA. P site: Peptidyl-tRNA site -

carries the growing polypeptide chain.

A site: Aminoacyl-tRNA site -holds the tRNA carrying the next AA to be added.

E site: Exit site

Translation Steps

1. Initiation

2. Elongation

3. Termination

Initiation

Brings together: mRNA A tRNA carrying the 1st AA 2 subunits of the ribosome

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

Initiation

Requires other proteins called "Initiation Factors”.

GTP used as energy source.

Elongation Steps:

1. Codon Recognition

2. Peptide Bond Formation

3. Translocation

Codon Recognition

tRNA anticodon matched to mRNA codon in the A site.

Peptide Bond Formation

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.

Translocation 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.

Comment

Elongation takes 60 milliseconds for each AA added.

Termination

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.

Let’s see Translation in motion…

http://www.hhmi.org/biointeractive/media/DNAi_translation_vo2-lg.mov

Polyribosomes

Cluster of ribosomes all reading the same mRNA.

Another way to make multiple copies of a protein.

Prokaryotes

Comment

Polypeptide usually needs to be modified before it becomes functional.

Examples

Sugars, lipids, phosphate groups added.

Some AAs removed. Protein may be cleaved. Join polypeptides together

(Quaternary Structure).

Signal Hypothesis

“Clue” on the growing polypeptide that causes ribosome to attach to ER.

All ribosomes are “free” ribosomes unless clued by the polypeptide to attach to the ER.

Result

Protein is made directly into the ER .

Protein targeted to desired location (e.g. secreted protein).

“Clue” (the first 20 AAs are removed by processing).

Mutations

Changes in the genetic makeup of a cell.

May be at chromosome or DNA level

Chromosome Alterations

Deletions Duplications Inversions Translocations

General Result

Loss of genetic information. Position effects: a gene's

expression is influenced by its location to other genes.

Evidence of Translocation

Translocations

Cri Du Chat Syndrome

Part of p arm of #5 has been deleted.

Good survival. Severe mental retardation. Small sized heads common.

Philadelphia Chromosome

An abnormal chromosome produced by a translocation of portions of chromosomes 9 and 22.

Causes chronic myeloid leukemia.

Mutation types - Cells

Somatic cells or body cells – not inherited

Germ Cells or gametes - inherited

DNA or Point Mutations

Changes in one or a few nucleotides in the genetic code.

Effects - none to fatal.

Types of Point Mutations

1. Base-Pair Substitutions

2. Insertions

3. Deletions

Base-Pair Substitution

The replacement of 1 pair of nucleotides by another pair.

Sickle Cell Anemia

Lets see how this mutation will affect the cell…

http://www.hhmi.org/biointeractive/media/DNAi_sicklecell-lg.mov

Types of Substitutions

1. Missense - altered codons, still code for AAs but not the right ones

2. Nonsense - changed codon becomes a stop codon.

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 Effect

Can be none to fatal depending on where the AA was in the protein.

Ex: if in an active site - major effect. If in another part of the enzyme - no effect.

Nonsense Effect

Stops protein synthesis. Leads to nonfunctional

proteins unless the mutation was near the very end of the polypeptide.

Sense Mutations

The changing of a stop codon to a reading codon.

Result - longer polypeptides which may not be functional.

Ex. “heavy” hemoglobin

Insertions & Deletions

The addition or loss of a base in the DNA.

Cause frame shifts and extensive missense, nonsense or sense mutations.

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 and the protein may still be able to function.

Mutagenesis

Process of causing mutations or changes in the DNA.

Mutagens

Materials that cause DNA changes.

1. Radiationex: UV light, X-rays

2. Chemicalsex: 5-bromouracil

Spontaneous Mutations

Random errors during DNA replication.

Comment

Any material that can chemically bond to DNA, or is chemically similar to the nitrogen bases, will often be a very strong mutagen.

Summary Know Beadle and Tatum. Know the central dogma. Be able to “read” the genetic

code. Be able to describe the events

of transcription and translation.

Summary

Be able to discuss RNA and protein processing.

Be able to describe and discuss mutations.