chapter 17 from gene to protein

Chapter 17From Gene to

Protein

Question?

How does DNA control a cell? By controlling Protein

Synthesis. Proteins are the link between

genotype and phenotype.

Central DogmaDNA

Transcription

RNA Translation

Polypeptide

Explanation

DNA - the Genetic code or genotype.

RNA - the message or instructions.

Polypeptide - the product for the phenotype.

DNA vs RNA

DNA RNASugar – deoxyribose riboseBases – ATGC AUGCBackbones – 2 1Size – very large smallUse – genetic code varied

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.

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.

Code Redundancy

Wobble effect: 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 tet 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.

TATA Box

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 Factors

Proteins that bind to DNA before RNA Polymerase.

Recognizes TATA box, attaches, and “flags” the spot for RNA Polymerase.

Initiation

Actual unwinding of DNA to start RNA synthesis.

Requires Initiation Factors.

Elongation

RNA Polymerase untwists DNA 1 turn at a time and adds complimentary bases.

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.

RNA Splicing

Removal of non-protein coding regions of RNA.

Coding regions are then spliced back together.

Introns

Intervening sequences. Removed from RNA. Some contain sequences that

regulate gene expression and many affect gene products

Exons

Expressed sequences of RNA.

Translated into AAs.

Introns - Function

Left-over DNA (?) Way to lengthen genetic

message to protect coding regions.

Old virus inserts (?)

Introns- Function

Way to create new proteins with exon shuffling

New combinations of exons= new proteins for evolution

Final RNA Transcript

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 tRNAYou have a diagram of this

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.

Is a compliment to the mRNA bases, so reads the same as the DNA codon.

Example

DNA - GAC mRNA - CUG tRNA anticodon - GAC

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.

Polyribosomes

Cluster of ribosomes all reading the same mRNA.

Another way to make multiple copies of a protein.

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).

End of Part 1

Mutations

Changes in the genetic makeup of a cell.

May be at chromosome (review chapter 15) or DNA level

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

Types of Substitutions

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

2. Nonsense - changed codon becomes a stop codon.

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.

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.

What is a gene?

A gene is a region of DNA that can be expressed to produce a final functional product.

The product can be a protein or a RNA molecule

Protein vs RNA

Protein – usually structure or enzyme for phenotype

RNA – often a regulatory molecule which will be discussed in future chapters.