Chapter 17

From Gene to Protein

• Describe the structure of DNA. What

is its elemental makeup? Name the

subunit that makes up DNA. What

components make up the DNA

molecule? How are the two strands

related and connected? How are they

different?

Question?

• How does DNA control a cell?

• By controlling Protein Synthesis.

• Proteins are the link between genotype

and phenotype.

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 Dogma

DNA

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

• Ex – AT, TA, CA, GC

• 42 = 16 amino acids

• Still not enough combinations.

If you use:

• 3 bases = 1AA

• Ex – CAT, AGC, TTT

• 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.

DNA vs RNA

DNA RNA

Sugar – deoxyribose ribose

Bases – ATGC AUGC

Backbones – 2 1

Size – very large small

Use – genetic code varied

Codon Dictionary

• Start- AUG (Met)

• Stop- UAA

UAG

UGA

• 60 codons for the other 19 AAs.

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.

TATA Box

• Short segment of T,A,T,A

• Located 25 nucleotides upstream for

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.

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.

Comment

• The head and tail areas often contain

“leaders” and “trailers”, areas of RNA

that are not read.

• Similar to leaders or trailers on

cassette tapes.

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.

snRNA

• Small Nuclear RNA.

• 150 nucleotides long.

• Structural part of spliceosomes.

snRNPs

• ("snurps")

• Small Nuclear Ribonucleoprotiens

• Made of snRNA and proteins.

• Join with other proteins to form a

spliceosome.

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

• If a segment of DNA is 5’-TGA AGA

CCG-3’, the resulting RNA strand

from the transcription of this would

read:

– 5’-TGA AGA CCG-3’

– 5’-UGA AGA CCG-3’

– 3’- CGG UCU UCA- 5’

– 3’- ACU UCU GGC- 5’

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.

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).

• The flow of genetic information from

DNA to protein in eukaryotic cells is

called the central dogma of biology.

– Write the central dogma of biology as a flow

chart

– What role does each of these structures play

in protein synthesis?

• DNA

• mRNA

• RNA polymerase

• Spliceosomes

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.

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”.

Comment

• Silent mutations may still have an

effect by slowing down the “speed” of

making the protein.

• Reason – harder to find some tRNAs

than others.

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. Radiation

ex: UV light, X-rays

2. Chemicals

ex: 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.

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.

• Be able to discuss “what is a gene”.