From Gene to Protein

From Gene to Protein

Chapter 17

I. Connection of Genes to Protein

A. Early Evidence - from the study of metabolic defects

1. Gerrod’s Hypothesis 1909

Archibald Gerrod - first to suggest that genes dictate phenotype through

enzymes.


1. Gerrod’s Hypothesis 1909

Studied Alkaptonuria - absence of an enzyme that breaks down

Alkapton

Symptoms of disease due to inability to make an enzyme

Enzymes needed for metabolic pathways


2. Beadle and Ephrussi, 1930s

Mutations affecting drosophila eye color

Blocks pigment by absence of a

pathway enzyme.

Mere hypothesis.


3. Beadle and Tatum

Finally established the link between genes and enzymes

Studied nutrition of bread mold

Neurospora crassa

(Study text for details)


4. One Gene - One Polypeptide Hypothesis

Refined from Beadle and Tatum

Not all proteins are enzymes

Specific proteins require specific genes

Used to be

One gene - one protein

Why the change?

B. The Process of Protein Synthesis - General Overview

1. Genes = instructions for a protein

2. RNA = bridge between DNA and protein

RNA distinctives

Single stranded

Ribose sugar

Uracil instead of Thymine


3. DNA nucleotide sequence

translates to Amino Acid sequence


4. Two Stages in Protein Synthesis

Transcription

DNA code to RNA copy

Messenger RNA (mRNA)

Translation

mRNA code

to Amino Acid sequence


5. Eukaryotes and Prokaryotes

Prokaryotes - no nucleus

Transcription and translation are

closely coupled

Eukaryotes

Transcription - in nucleus

RNA processing - in nucleus

Translation - cytoplasmic ribosome


6. Pathway summary

DNA - RNA - Protein

C. Structure of the Genetic Code

1. Triplets of nucleotides specify amino acids

3 letters = dictate one amino acid

With 4 letters = 64 triplet combinations

2. Each Triplet called a Codon


3. Matching codons to their amino acids began in the 1960s

First - Marshall Nirenberg

Mid 1960s - completed

61 code for amino acids

AUG = start of a gene

3 codons = termination of a gene


4. Redundant, but not ambiguous

Several codons can code for 1 amino acid

(redundant)

One codon can mean only 1 amino acid

(not ambiguous)

II. Transcription and RNA

A. DNA in the nucleus

but Proteins built in cytoplasm

Need an RNA copy of the gene to take

code to the cytoplasm.

Transcription - making the RNA copy


B. RNA Polymerase - opens up the specific gene and adds RNA nucleotides according to the DNA sequence.

Prokaryotes - 1 kind RNA polymerase

Eukaryotes - RNA Polymerases I, II, III

C. Gene is read 3’ to 5’

RNA built 5’ to 3’


D. Beginning and end of gene

Promotor sequence begins

Terminator signals the end


E. Three stages in Transcription

Initiation

Elongation

Termination

II. Transcription and RNA F. Initiation

Promotor sequence

start of a gene

determines which strand is template

provides a binding site for

RNA Polymerase

II. Transcription and RNA F. Initiation

In Eukaryotes

Transcription factors find promotor

(Often a TATA sequence)

Bind to promotor

RNA Polymerase binds onto Trans Fact

makes Transcription Initiation Complex

II. Transcription and RNA G. Elongation

RNA Polymerase moves down DNA

Untwists and unzips DNA (10-20 bases)

Adds in RNA nucleotides to 3’ end of

growing RNA chain

Behind this - DNA connects back and twists

RNA copy peels away.

II. Transcription and RNA G. Elongation

Many RNA Polymerases can be making a mRNA copy of the same gene at the same time.

= More mRNAs

increases the speed of protein production

II. Transcription and RNA H. Termination

At end, RNA Polymerase copies a

terminator

In prokaryotes

Transcription ends at end of terminator

In Eukaryotes

Transcription continues far beyond it

This longer “pre-RNA” is then released

I. Modification of mRNA after transcription

Enzymes modify the “pre-RNA” before its sent out to make protein

1. At 5’ end, a modified Guanine is added

the 5’ Cap

Function?

Prevents erosion

“Attach here” signal for

ribosomes


Enzymes modify the “pre-RNA” before its sent out to make protein

2. At 3’ end - Poly A tail - 50 -250 letters

Protects against hydrolysis damage

Facilitates ribosome attachment

Facilitates export from nucleus


3. RNA Splicing

mRNA has long stretches of non-coding nucleotides in between codes

Called Introns

Coding regions called Exons

Splicing removes many intron portions


3. RNA Splicing

Accomplished by Spliceosome

Composed of;

Several proteins

snRNP - small nuclear

Ribonuclearproteins

each snRNP has a RNA

molecule, 150 letters


3. RNA Splicing

Functions

1.Contol which introns stay in

2.Regulate passage out of nucleus

3.Splice genes to code for more than

one polypeptide


3. RNA Splicing

Alternative RNA Splicing

More than one polypeptide from one

gene.

Depends on which segments are treated as exons


4. Split genes facilitates protein evolutionProteins have different regions - domainsdifferent exons may code for different

domains

Presence of introns - facilitate crossing over at that point.

Creates new genes by mixing exons from different genes.

Leads to new proteins

III. Translation A. mRNA sequence to protein

Transfer RNAs place amino acids in correct order according to the

sequence of codons

III. Translation B. Transfer RNA - tRNA

Made according to genes in the nucleus

Can be used in the cytoplasm over and over

1. Action - picks up a specific amino acid

places it in correct sequence at ribosome

Returns to cytosol to get another of the same amino acid


2. Structure of tRNA

80 nucleotides

Loops back on itself for a 3D shape

At one end - attachment for a specific amino acid.

At opposite end - Anticodon

compliments and binds to the codon

for the amino acid it carries


3. Some anticodons recognize more than one codon.Why? Two reasons

- The third base in a codon includes some flexibility- called Wobble

At this Wobble position

U on anticodon can bind to

A or G in third codon position

- Some tRNA anticodons have Inosine

A modified Adenine

Can bond with U,C, or A.


4. Amino Acid joins to tRNA by

Aminoacyl-tRNA Synthetase

20 different Synthetases to match

the 20 different amino acids

Each has active sites for a specific

tRNA and A. Acid combo.

Forms a covalent bond.

III. Translation C. Ribosomes

Composed of large and small subunit

made of proteins and rRNA (Active parts)

Subunits form in the nucleolus

exit nucleus through nuclear pores

Large and small subunits only joins as a functional ribosome when they contact a mRNA molecule

Prokaryotic ribosomes differ from eukaryotic ones

Ribosome Structure

III. Translation C. Ribosomes

Binding Sites

P - tRNA with chain

A - tRNA with next

A. Acid

E - Discharges used

tRNAs.

III. Translation D. Details of the Process

1. Initiation

2. Elongation

3. Termination

All need protein aids

1 and 2 need energy

hydrolysis of GTP

D. Details of the Process

1. Initiation - Brings together

mRNA

First tRNA with A.Acid

Two Ribosome subunits


1. Initiation

Sm. Ribosomal subunit

binds mRNA

and initiator tRNA (methionine)

Attaches to first codon


1. Initiation

Initiation factors bring Lg subunit

Attaches so that

Initiator tRNA occupies P site


2. Elongation

Series of 3 step cycles

Codon Recognition

A tRNA binds to mRNA codon

at the A site

Binding assisted by an

Elongation factor

Requires hydolysis of 2 GTPs


2. Elongation


Peptide Bond Formation

new A. acid in A site is bonded

to growing chain at P site

Peptide bond catalyzed by

rRNA

tRNA at P site separates from

chain

Chain now hooked to A site

2. Elongation


Translocation

Ribosome moves the tRNA

at A site with chain

To the P site

This slides the mRNA in

(anti-codon still bonded)

tRNA from P site moves to E

and leaves ribosome

Fueled by hydrolysis of GTP

3. Termination

When a stop codon reaches A site

Release factor binds to stop codon

breaks the bond (hydrolyzes)

between the tRNA and chain at the

P site

Polypeptide chain is now free

Translation complex disassembles

E. Many polypeptides can be made from a mRNA at the same time.

More than one ribosome can travel down a mRNA at the same time

Polyribosomes

F. After translation

Polypeptide folds up spontaneously

Primary to ….. Tertiary … quaternary

G. Post-translational modifications

1. Additions of

sugars, lipids, phosphate groups

2. Enzymes may cleave the chains or remove sections

3. Multiple polypetides may join

Tertiary structures to Quaternary

H. Post-translational destinations of proteins

Final destinations for proteins effects how they are processed during translation

1. Two kinds of Ribosomes

Free - suspended in cytosol

translate proteins for cytosol

Bound - Attached to cytosol side

of Endoplasmic Reticulum

Synthesizes proteins for

1. The endomembrane system

2. Secretion outside cell


2. All Ribosomes begin translating in cytosol

But

Whether the protein will be

1. Part of endomembrane system

or

2. For export out of cell

It has a Signal Peptide region at the leading end (synthesized first)

About 20 Amino acids


Signal Recognition Particle (SRP)

Binds to the Signal Peptide

Attaches it and its Ribosome to the ER

SRP - Protein RNA combo

After binding, SRP leaves


Growing protein snakes through protein pore in ER membrane into cisternal space

When complete

An enzyme cleaves off the signal peptide

Secretory proteins gather completely in cisternal space

ER Membrane proteins remain embedded


3. Other kinds of Signal Peptides send proteins to

Mitochondria, Chloroplasts, Nucleus, other organelles

Acts like a postal code

Translation is completed in cytosol by free ribosomes

IV. RNA RolesA. Diverse functions based on

1. Ability of RNA to form H-bonds with other nucleic acids

2. Ability to take on a 3D shape by looping back on itself with H-bonds

(tRNA)

B. RNA more versatile than DNA

Structural

Informational

Catalytic

V. Prokaryote vs. EukaryoteA. Eukaryotic RNA polymerases differ

B. Eukaryotic RNA polymerases require transcription factors

C. Transcription termination differs

D. Ribosomes differ

E. Prokaryotes can transcribe and translate a gene at the same time

Eukaryotes, nuclear membrane separates the two processes

Additional steps needed for coordination

VI. Mutations

A. Change in one base pair = Point Mutation

1. In gametes? - to future generations

2. Example - Sickle-cell Anemia

3. Base-pair substitution

Replacement of one pair with

another

VI. MutationsA. Change in one base pair = Point Mutation


Silent mutations - no effect, why?

- change results in same amino acid due to redundancy in code

- change results in new amino acid with same properties

- change in protein region where amino acid sequence is not essential

VI. Mutations

A. Change in one base pair = Point Mutation


Some cause key changes

Sometimes advantageous

At protein active sites - more likely to have impact

VI. Mutations

B. Missense mutations - still code for an amino acid, but for a different one

C. Nonsense mutations - change an amino acid codon to a stop codon.

D. Insertions or Deletions

Disastrous effect

Frameshift - All nucleotides downstream will be improperly grouped into codons

VI. Mutations

E. Causes of Mutations

Country Music!

1. Errors occuring during

DNA Replication

DNA Repair

DNA Recombination

Called spontaneous mutations

VI. Mutations

E. Causes of Mutations

2. Mutagens - Chemicals or physical agents that alter DNA

(Country Music?)

Physical agents - radiation

VI. MutationsE. Causes of Mutations

2. Mutagens - Chemicals or physical agents that alter DNA

Chemical agent methods

- Some mimic DNA bases

causes incorrect pairing

- Insertion into and distortion of the double helix

- Chemical changes to bases

Connection mutagens - carcinogens

How do we define Gene ?

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From Gene to Protein