Chapter 17

From Gene to Protein Rob Swatski

Associate Professor of Biology

HACC – York Campus

Overview: The Flow of Genetic Information

DNA information = specific sequences of nucleotides

DNA protein synthesis

Proteins: link genotype & phenotype

Gene expression: DNA directs protein synthesis

- 2 stages: transcription & translation

How was the fundamental relationship between genes & proteins discovered?

- examine evidence from studies of

metabolic defects

1909: British physician Archibald Garrod 1st suggested that genes dictate phenotypes with enzymes

- symptoms of inherited disease reflects inability to synthesize a certain enzyme

- required understanding that cells synthesize & degrade molecules using metabolic pathways

Neurospora bread mold

Nutritional Mutants in Bread Mold

George Beadle & Edward Tatum exposed Neurospora to x-rays

- created mutants that could not survive on minimal medium

(cannot synthesize certain molecules)

Identified 3 classes of arginine-deficient mutants

- each lacked a different enzyme needed to make arginine

Developed the “one gene – one enzyme hypothesis”

- each gene directs the synthesis of a specific enzyme

Minimal medium

No growth: Mutant cells cannot grow and divide

Growth: Wild-type cells growing and dividing

EXPERIMENT

RESULTS

Classes of Neurospora crassa

Wild type Class I mutants Class II mutants Class III mutants

Minimal medium (MM) (control)

MM ornithine

MM citrulline

Co

nd

itio

n

MM arginine (control)

Summary of results

Can grow with or without any supplements

Can grow on ornithine, citrulline, or arginine

Can grow only on citrulline or arginine

Require arginine to grow

Growth No growth

CONCLUSION

Wild type

Class I mutants (mutation in

gene A)

Class II mutants (mutation in

gene B)

Class III mutants (mutation in

gene C)

Gene (codes for enzyme)

Gene A

Gene B

Gene C

Precursor Precursor Precursor Precursor

Enzyme A Enzyme A Enzyme A Enzyme A

Enzyme B Enzyme B Enzyme B Enzyme B

Ornithine Ornithine Ornithine Ornithine

Enzyme C Enzyme C Enzyme C Enzyme C

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

The Products of Gene Expression: A Developing Story

• Some proteins aren’t enzymes… so researchers later revised the hypothesis to “one gene – one protein”

• But, many proteins consist of several polypeptides, each having its own gene

Beadle & Tatum’s hypothesis is now restated as the:

“one gene–one polypeptide hypothesis”

Basics of Transcription & Translation

RNA: the bridge between genes & the proteins they code

• Transcription: synthesis of RNA under the direction of DNA

- produces messenger RNA (mRNA)

• Translation: synthesis of a polypeptide under the direction of mRNA

- ribosomes: the sites of translation

Eukaryotes:

the nuclear envelope separates transcription from translation

- eukaryotic RNA transcripts are modified via RNA processing to yield finished mRNA

Prokaryotes:

mRNA transcripts are immediately translated without further processing

(a) Prokaryotic Cell (Bacteria)

TRANSCRIPTION DNA

mRNA

TRANSLATION Ribosome

Polypeptide

Primary transcript: initial RNA transcript from a gene before processing

Central dogma:

cells are governed by a cellular chain of command:

DNA RNA Protein

(b) Eukaryotic cell

TRANSCRIPTION

Nuclear envelope

DNA

Pre-mRNA RNA PROCESSING

mRNA

(b) Eukaryotic cell

TRANSCRIPTION

Nuclear envelope

DNA

Pre-mRNA RNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

The Genetic Code

How are the instructions for assembling amino acids into proteins encoded into DNA?

There are 20 amino acids,

… but there are only 4 nucleotide bases in DNA

How many bases correspond to an amino acid?

Codons: Triplets of Nucleotides

The flow of information from gene protein is based on a triplet code

- series of non-overlapping 3-nucleotide “words”

Triplet: smallest unit that can code for amino acids

- “AGT” = placement of serine at its correct position in the polypeptide

Transcription: one of the two DNA strands (template strand)

provides a pattern for ordering the nucleotide sequence in the mRNA transcript

Translation:

mRNA base triplets (codons) are read in the 5 to 3 direction

- each codon specifies the amino acid (1 of 20) and it’s correct position in a polypeptide

DNA template strand

TRANSCRIPTION

mRNA

TRANSLATION

Protein

Amino acid

Codon

Trp Phe Gly

5

5

Ser

U U U U U 3

3

5 3

G

G

G G C C

T

C

A

A

A A A A A

T T T T

T

G

G G G

C C C G G

DNA molecule

Gene 1

Gene 2

Gene 3

C C

Cracking the Code

• All 64 codons were deciphered by the mid-1960s

• Of the 64 triplets, 61 code for amino acids

- 3 triplets are stop codons that end translation:

UAA, UAG, UGA

• The genetic code is redundant but not ambiguous

• Codons must be read in the correct reading frame (correct groupings) in order to synthesize the specified polypeptide

Second mRNA base

Firs

t m

RN

A b

ase

(5 e

nd

of

cod

on

)

Thir

d m

RN

A b

ase

(3

en

d o

f co

do

n)

UUU

UUC

UUA

CUU

CUC

CUA

CUG

Phe

Leu

Leu

Ile

UCU

UCC

UCA

UCG

Ser

CCU

CCC

CCA

CCG

UAU

UAC Tyr

Pro

Thr

UAA Stop

UAG Stop

UGA Stop

UGU

UGC Cys

UGG Trp

G C

U

U

C

A

U

U

C

C

C A

U

A

A

A

G

G

His

Gln

Asn

Lys

Asp

CAU CGU

CAC

CAA

CAG

CGC

CGA

CGG

G

AUU

AUC

AUA

ACU

ACC

ACA

AAU

AAC

AAA

AGU

AGC

AGA

Arg

Ser

Arg

Gly

ACG AUG AAG AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

Val Ala

GGU

GGC

GGA

GGG Glu

Gly

G

U

C

A

Met or start

UUG

G

Evolution of the Genetic Code

The genetic code is shared by all living things

- Genes can be transcribed & translated after being transplanted from one species to another

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

Synthesis of an RNA Transcript

The 3 Stages of Transcription:

1. Initiation

2. Elongation

3. Termination

Transcription: DNA RNA

RNA synthesis is catalyzed by RNA polymerase

- pries DNA strands apart

- hooks RNA nucleotides together

The RNA is complementary to the DNA template strand

Follows same base-pairing rules as DNA

- except uracil substitutes for thymine

Promoter: DNA sequence that RNA polymerase attaches to

Transcription unit: section of DNA that is transcribed

Promoter Transcription unit

DNA Start point

RNA polymerase

5 5 3

3

Initiation

3 3

1

RNA transcript

5 5

Unwound DNA

Template strand of DNA

2 Elongation Rewound DNA

5

5 5 3 3 3

RNA transcript

3 Termination 5

5

5 3 3

3

Completed RNA transcript

RNA Polymerase Binding & Initiation of Transcription

Promoters: signal initiation of RNA synthesis

- TATA box promoter is crucial in forming the initiation complex in eukaryotes

Transcription factors:

needed to help bind RNA polymerase & initiate transcription

Transcription initiation complex:

completed assembly of transcription factors & RNA polymerase bound to a promoter

Transcription initiation complex forms 3

DNA Promoter

Nontemplate strand

5 3

5 3

5 3

Transcription factors

RNA polymerase II

Transcription factors

5 3

5 3

5 3

RNA transcript

Transcription initiation complex

5 3

TATA box

T

T T T T T

A A A A A

A A

T

Several transcription factors bind to DNA 2

A eukaryotic promoter 1

Start point Template strand

Elongation of the RNA Strand

As RNA polymerase moves along DNA, it untwists the double helix, 10-20 bases at a time

- transcription rate = 40 nucleotides/sec

A gene can be transcribed simultaneously by several RNA polymerases

Nucleotides are added to the 3’ end of the growing RNA molecule

Elongation

RNA polymerase

Non-template strand of DNA

RNA nucleotides

3 end

Direction of transcription

(“downstream”) Template strand of DNA

Newly made RNA

3

5

5

Termination of Transcription

In bacteria:

RNA polymerase stops transcription at end of the terminator & the mRNA can be translated

without further modification

In eukaryotes:

RNA polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA

chain

- polymerase eventually falls off the DNA

Eukaryotic Cells Modify RNA After Transcription

Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before mRNA “gene” enters

cytoplasm

Both ends of the primary transcript are usually altered

- and some interior parts of RNA are usually cut-out & other parts spliced together

Alteration of mRNA Ends

Each end of pre-mRNA is modified in a particular way:

- the 5 end gets a modified nucleotide 5 cap

- the 3 end gets a poly-A tail

Why Modify? - Facilitates export of mRNA

- Protects mRNA from hydrolytic enzymes

- Helps ribosomes attach to the 5 end

Protein-coding segment

Polyadenylation signal

5 3

3 5 5 Cap UTR Start codon

G P P P

Stop codon

UTR

AAUAAA

Poly-A tail

AAA AAA …

Split Genes & RNA Splicing

Most genes & their RNA transcripts have long noncoding regions (introns) that lie between

coding regions

- intron = intervening sequences (“in the way”)

Exons: coding regions

- expressed & translated into amino acid sequences

RNA splicing: removes introns & joins exons

- creates mRNA molecule with a continuous coding sequence

5 Exon Intron Exon

5 Cap Pre-mRNA Codon numbers

130 31104

mRNA 5 Cap

5

Intron Exon

3 UTR

Introns cut out and exons spliced together

3

105 146

Poly-A tail

Coding segment

Poly-A tail

UTR 1146

Some RNA splicing is carried out by spliceosomes

- consist of a variety of proteins & small nuclear ribonucleoproteins (snRNPs = “snurps”)

- snRNPs can recognize the splice sites

RNA transcript (pre-mRNA)

Exon 1 Exon 2 Intron

Protein

snRNA

snRNPs

Other proteins

5

5

Spliceosome

Spliceosome components

Cut-out intron mRNA

Exon 1 Exon 2 5

Ribozymes Catalytic RNA molecules that act as enzymes & splice RNA

- not all biological catalysts are proteins!

How can RNA function as an enzyme?

- can form a 3-D structure because it can base-pair with itself

- some RNA bases contain functional groups

- can hydrogen-bond with other nucleic acids

Some genes can encode more than 1 kind of polypeptide, depending on which segments are

treated as exons during RNA splicing

- the actual # of different proteins an organism can produce is much greater than its number of genes

Alternative RNA Splicing

Proteins often have a modular architecture consisting of discrete regions called domains

- different exons can code for different domains in a protein

Exon shuffling can result in the evolution of new proteins

Gene

DNA Exon 1 Exon 2 Exon 3 Intron Intron

Transcription

RNA processing

Translation

Domain 2

Domain 3

Domain 1

Polypeptide

Molecular Components of Translation

A cell translates mRNA message into protein with the help of transfer RNA (tRNA)

tRNA molecules are not identical:

- each carries a specific amino acid on one end

- each has an anticodon on the other end that base-pairs with a complementary codon on mRNA

Polypeptide

Ribosome

Amino acids

tRNA with amino acid attached

tRNA

Anticodon

Codons 3 5

mRNA

The Structure & Function of tRNA

tRNA: one RNA

strand, 80 nucleotides long

- when flattened, it resembles a cloverleaf

C

Amino acid attachment site

Hydrogen bonds

Anticodon

3

5

Can twist & fold into an “L”-shaped 3-D molecule through hydrogen-bonding

Amino acid attachment site

3

3

5

5

Hydrogen bonds

Anticodon Anticodon

(b) 3-D structure (c) Symbol used in this book

Translation requires 2 steps: “The Match Game”

1. tRNA and its amino acid are matched by the enzyme aminoacyl-tRNA synthetase

- forms “charged tRNA”

2. tRNA anticodon and an mRNA codon are matched

Flexible pairing at the 3rd base of a codon is called wobble

- allows some tRNAs to bind to more than 1 codon

Aminoacyl-tRNA synthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

P P i

i

i

Adenosine

tRNA

Adenosine P

tRNA

AMP

Computer model

Amino acid

Aminoacyl-tRNA synthetase

Aminoacyl tRNA (“charged tRNA”)

Ribosomes

Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis

- 2 ribosomal subunits (large & small) are made of proteins & ribosomal RNA (rRNA)

Growing polypeptide

Exit tunnel tRNA molecules

Large subunit

Small subunit

(a) Computer model of functioning ribosome

mRNA

E P A

5 3

Exit tunnel

A site (Aminoacyl- tRNA binding site)

Small subunit

Large subunit

P A

P site (Peptidyl-tRNA binding site)

mRNA binding site

(b) Schematic model showing binding sites

E site (Exit site)

E

A ribosome has 3 binding sites for tRNA:

- A site: holds the tRNA carrying the next amino acid to be

added to the chain

- P site: holds the tRNA carrying the growing polypeptide

chain

- E site (Exit): where discharged tRNAs leave the ribosome

Amino end

mRNA E

(c) Schematic model with mRNA and tRNA

5 Codons

3

tRNA

Growing polypeptide

Next amino acid to be added to polypeptide chain

Building a Polypeptide

The 3 stages of translation:

1. Initiation

2. Elongation

3. Termination

All 3 stages require protein factors

Initiation of Translation

Initiation stage: brings together mRNA, a tRNA with the 1st amino acid, & the 2 ribosomal subunits

1. First, the small ribosomal subunit binds with mRNA and a special initiator tRNA

2. Then the small subunit moves along mRNA until it reaches the start codon (AUG)

- Initiation factors bring in the large subunit to complete the translation initiation complex

3

3 5 5 U

U A

A C

G

GTP GDP Initiator

tRNA

mRNA

5 3 Start codon

mRNA binding site

Small ribosomal subunit

5

P site

Translation initiation complex

3

E A

Large ribosomal subunit

Elongation of the Polypeptide Chain

Elongation stage: amino acids are added one by one

Each addition involves elongation factors and occurs in 3 steps:

a. Codon recognition

b. Peptide bond formation

c. Translocation

Amino end of polypeptide

mRNA

5

3 E

P site

A site

GTP

GDP

E

P A

Amino end of polypeptide

mRNA

5

3 E

P site

A site

GTP

GDP

E

P A

E

P A

Amino end of polypeptide

mRNA

5

3 E

P site

A site

GTP

GDP

E

P A

E

P A

GDP

GTP

Ribosome ready for next aminoacyl tRNA

E

P A

Termination of Translation

Termination:

- occurs when a stop codon in mRNA reaches the A site

The A site accepts a release factor protein

- adds a water molecule instead of an amino acid

- this releases the polypeptide

- the translation complex comes apart

Release factor

3

5

Stop codon (UAG, UAA, or UGA)

5

3

2

Free polypeptide

2 GDP

GTP

5

3

Polyribosomes

Groups of ribosomes that simultaneously translate one mRNA, forming a polyribosome (polysome)

- allows a cell to quickly make many copies of a polypeptide

Growing polypeptides

Completed polypeptide

Incoming ribosomal subunits

Start of mRNA

(5 end) End of mRNA

(3 end) (a)

Ribosomes

mRNA

(b) 0.1 µm

Post-Translation

A protein is usually not functional immediately after translation

- requires further post-translational modification

It spontaneously coils and folds into its correct 3-D shape

- some activated by enzymes that cleave them

- others assemble into protein subunits

Targeting Polypeptides to Specific Locations

Two populations of ribosomes are found in cells:

- Free ribosomes: synthesize proteins that function in the cytosol

- Bound ribosomes: synthesize proteins on ER and those that will be secreted from the cell

Ribosomes are identical and can switch from free to bound

Polypeptide synthesis always begins and ends in the cytosol

- unless the polypeptide signals the ribosome to attach to the ER

Polypeptides destined for the ER or for secretion are marked by a signal peptide

- a signal-recognition particle (SRP) binds to the signal peptide

- the SRP brings the signal peptide & its ribosome to the ER

Ribosome

mRNA

Signal peptide

Signal- recognition particle (SRP)

CYTOSOL Translocation complex

SRP receptor protein

ER LUMEN

Signal peptide removed

ER membrane

Protein

Point Mutations - chemical changes in just 1 base pair of a gene

- a change in one DNA nucleotide can lead to the production of an abnormal protein

Wild-type hemoglobin DNA

mRNA

Mutant hemoglobin DNA

mRNA

3

3

3

3

3

3

5

5

5

5

5

5

C C T T T

T G G A A A

A

A A A G G U

Normal hemoglobin Sickle-cell hemoglobin

Glu Val

Types of Point Mutations

- Base-pair substitutions

- Base-pair insertions or deletions

Wild type

3 DNA template strand

3

3 5

5

5 mRNA

Protein

Amino end

Stop

Carboxyl end

A instead of G

3

3

3

U instead of C

5

5

5

Stop

Silent mutations: have no effect on the amino acid because of redundancy in the genetic code

Wild type

DNA template strand

3

5

mRNA

Protein

5

Amino end

Stop

Carboxyl end

5

3

3

T instead of C

A instead of G

3

3

3

5

5

5

Stop

Missense: still codes for an amino acid, but not necessarily the right amino acid

Wild type

DNA template strand

3

5

mRNA

Protein

5

Amino end

Stop

Carboxyl end

5

3

3

A instead of T

U instead of A

3

3

3

5

5

5

Stop

Nonsense: changes an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

Insertions and Deletions

- additions or losses of nucleotide pairs in a gene

Can have a disastrous effect on the protein more often than substitutions

- may produce a frameshift mutation, which alters the reading frame

Wild type

DNA template strand

3

5

mRNA

Protein

5

Amino end

Stop

Carboxyl end

5

3

3

Extra A

Extra U

3

3

3

5

5

5

Stop

Frameshift Mutation causing immediate nonsense (1 base-pair insertion)

Wild type

DNA template strand

3

5

mRNA

Protein

5

Amino end

Stop

Carboxyl end

5

3

3

missing

missing

3

3

3

5

5

5

Frameshift Mutation causing extensive missense (1 base-pair deletion)

Wild type

DNA template strand

3

5

mRNA

Protein

5

Amino end

Stop

Carboxyl end

5

3

3

missing

missing

3

3

3

5

5

5

No Frameshift Mutation, but one amino acid missing (3 base-pair deletion)

Stop

What Is a Gene?

We have considered a gene as:

- A discrete unit of inheritance

- A region of specific nucleotide sequence in a chromosome

- A DNA sequence that codes for a specific polypeptide chain

In summary, a gene can be defined as:

- a region of DNA that can be expressed to

produce a final functional product, either a

polypeptide or an RNA molecule

TRANSCRIPTION

RNA PROCESSING

DNA

RNA transcript

3

5 RNA polymerase

RNA transcript (pre-mRNA)

Intron

Exon

NUCLEUS

Aminoacyl-tRNA synthetase

AMINO ACID ACTIVATION

Amino acid

tRNA CYTOPLASM

Growing polypeptide

3

Activated amino acid

mRNA

TRANSLATION

Ribosomal subunits

5

E

P

A

A Anticodon

Ribosome

Codon

E