Chapter 17

From Gene to Protein

How an Organism’s GenotypeProduces Its Phenotype

The information content of DNA is in the form of specific sequences of nucleotides

The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

Proteins are links between genotype and phenotype

DNA RNA Protein

• Transcription

– Is the synthesis of RNA under the direction of DNA– Produces messenger RNA (mRNA)

• Translation

– Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA

– Occurs on ribosomes

Transcription Translation

Gene expression- process by which DNA directs protein synthesis

Minimal medium

No growth:Mutant cellscannot growand divide

Growth:Wild-typecells growingand dividing

EXPERIMENT Classes of Neurospora crassa

Wild type Class I mutants Class II mutants Class III mutants

Minimalmedium(MM)(control)

MM ornithine

MM citrulline

Co

nd

itio

n

MM arginine(control)

Summaryof results

Can grow withor without anysupplements

Can grow onornithine,citrulline, orarginine

Can grow onlyon citrulline orarginine

Require arginineto grow

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 forenzyme)

Gene A

Gene B

Gene C

Precursor Precursor Precursor PrecursorEnzyme A Enzyme A Enzyme A Enzyme A

Enzyme B Enzyme B Enzyme B Enzyme B

Enzyme C Enzyme C Enzyme C Enzyme C

Ornithine Ornithine Ornithine Ornithine

Citrulline Citrulline Citrulline Citrulline

Arginine Arginine Arginine Arginine

• George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules

• Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine

Beadle, Tatum, and colleagues-

one gene- one enzyme

states that each gene dictates production of a specific enzyme

one gene- one proteinresearchers revised since some proteins aren’t enzymes

one gene–one polypeptide hypothesis-

states that the function of an individual gene is to dictate the

production of a specific polypeptide

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

TRANSCRIPTIONDNA

mRNA

(a) Bacterial cell

TRANSLATION

Ribosome

Polypeptide

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Prokaryotic cell

Nuclearenvelope

TRANSCRIPTION

Eukaryotic cell

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Pre-mRNA

Prokaryotic cell

Nuclearenvelope

mRNA

TRANSCRIPTION

RNA PROCESSING

Eukaryotic cell

TRANSCRIPTION

TRANSLATION

DNA

mRNA

Ribosome

Polypeptide

DNA

Pre-mRNA

Prokaryotic cell

Nuclearenvelope

mRNA

TRANSLATION

TRANSCRIPTION

RNA PROCESSING

Ribosome

Polypeptide

Eukaryotic cell

In prokaryotes, mRNA produced by transcription is immediately translated without more processing

In a eukaryotic cell, the nuclear envelope separates transcription from translation

Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

primary transcript

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 four nucleotide bases in DNA

• So how many bases correspond to an amino acid?

• The flow of information from gene to protein is based on a

triplet code

a series of nonoverlapping, three-nucleotide words

• During transcription, a DNA strand called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript

• The template strand is always the same strand for a given gene

• During translation, the mRNA base triplets, codons,

are read in the 5 to 3 direction

• Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

“coding strand”

Genetic Code Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

• 64 codons – includes 3 Stop codons

• redundant (>1 codon may specify a particular aa) but not ambiguous; no codon specifies more than 1 aa

• codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced

Evolution of the Genetic Code

• The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals

• Genes can be transcribed and translated after being transplanted from one species to another

(a) Tobacco plant expressing a firefly gene

(b) Pig expressing a jellyfish gene

April 30, 2012

With apologies to the Smashing Pumpkins

Announcements

• Final exam: Friday, May 11 6-8 PM– Written study guide tomorrow– MC practice exam by Friday

• Quiz over 16 and 17 due Tuesday May 1

• MB homework 8: Ch 18 due May 3

Ch 17: from gene to protein

• Understanding the link between genes and polypeptides

• The genetic code

• Transcription

• Translation

• Mutations

Molecular Components of Transcription

• RNA synthesis

– is catalyzed by RNA polymerase,

which pries the DNA strands apart & hooks together RNA nucleotides

– follows the same base-pairing rules as DNA, except uracil substitutes for thymine

the stretch of DNA that is transcribed

the DNA sequence where RNA polymerase attaches

Promoter

35

Transcription unit

DNA

InitiationRNA polymerase

Start point

Template strandof DNA

RNAtran-script

UnwoundDNA

Elongation

3

3

53

5

5

3 5

RewoundDNA

5 3

35 35

RNAtranscript Termination

35

5 3Completed RNA transcript

After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand.

The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5 3. During transcription, the DNA strands re-form a double helix.

Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.

• Promoters signal the initiation of RNA synthesis

– A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

• Transcription factors (TFs; think cell communication) mediate the binding of RNA polymerase and initiation of transcription

• transcription initiation complex-

completed assembly of TFs & RNA polymerase II bound to a promoter

Promoter

53

35

TATA box Start point

Transcriptionfactors

53

35

Several transcriptionfactors

Additional transcriptionfactors

RNA polymerase IITranscription factors

RNA transcript

53

355

Transcription initiation complex

Eukaryotic promoter

TemplateDNA strand

Initiation

Elongation of the RNA Strand

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

• RNA polymerase adds nucleotides to the 3’ end of the growing RNA molecule as it continues along the double helix

• Transcription progresses at a rate of 40 nucleotides/ sec in eukaryotes

• A gene can be transcribed simultaneously by several RNA polymerases

Elongation Non-templatestrand of DNA

RNApolymerase

RNA nucleotides

3 end3

5

5

Newly madeRNA

Templatestrand of DNA

Direction of transcription(“downstream”)

“coding strand”

Termination of Transcription

• The mechanisms of termination are different in prokaryotes and eukaryotes

– In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification

– In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence

Eukaryotic cells

• Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm

• During RNA processing:

1. both ends of primary RNA transcript (pre-mRNA) are usually altered

2. usually some interior parts of the molecule are cut out, and the other parts spliced together

Alteration of mRNA EndsEach end of a pre-mRNA molecule is modified in a particular way:

1. The 5 end receives a modified nucleotide cap

2. The 3 end gets a poly-A tail

5Protein-coding segment

5 Start codon Stop codon Poly-A tail

Polyadenylation signal

5 3Cap UTR UTR

These modifications share several functions:1. They seem to facilitate the export of mRNA2. They protect mRNA from hydrolytic enzymes3. They help ribosomes attach to the 5’ end

5 Exon Intron Exon Intron Exon 3Pre-mRNA

1 30 31 104 105 146

Codingsegment

Introns cut out andexons spliced together

1 146

5Cap

5Cap

Poly-A tail

Poly-A tail

5 3UTR UTR

Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions

RNA splicing

• introns-

noncoding regions, intervening sequences

• exons-

expressed, usually translated into amino acid sequences

RNA splicing removes introns and joins exons,

creating an mRNA molecule w/ a continuous coding sequence

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

ProteinsnRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintronmRNA

Exon 1 Exon 25

• In some cases, RNA splicing is carried out by

spliceosomes

– recognize the splice sites– consist of:

• small nuclear ribonucleoproteins

(snRNPs) • a variety of proteins

Ribozymes

• Ribozymes-

are catalytic RNA molecules that function as enzymes and can splice RNA

• The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

The Functional & Evolutionary Importance of Introns

• Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing

• Such variations are called alternative RNA splicing

– Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes

• Proteins often have a modular architecture– consisting of discrete

structural and functional regions called domains

• In many cases- different exons code for the different domains in a protein

GeneDNA

Exon 1 Intron Exon 2 Intron Exon 3

Transcription

RNA processing

Translation

Domain 3

Domain 1

Domain 2

Polypeptide

Polypeptide

Ribosome

Aminoacids

tRNA withamino acidattached

tRNA

Anticodon

Trp

Phe Gly

Codons 35

mRNA

Molecular Components of Translation

A cell translates an mRNA message

into protein with the help of

transfer RNA (tRNA)

tRNA carry 2 things- one on each end:

1. specific amino acid

2. an anticodon

The anticodon base-pairs with acomplementary codon on mRNA

Amino acidattachment site

Hydrogenbonds

3

5

Two-dimensional structure

Anticodon

Amino acidattachment site

35

Hydrogenbonds

Anticodon Anticodon

Symbol used in this bookThree-dimensional structure

3 5

A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long

Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf

Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule

tRNA is roughly L-shaped

3 5

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Computer model

Aminoacid

Aminoacyl-tRNAsynthetase

Aminoacyl-tRNAsynthetase (enzyme)

Amino acid

P P P Adenosine

ATP

P

P

P

PPi

i

i

Adenosine

tRNA

AdenosineP

tRNA

AMP

Aminoacid

Aminoacyl tRNA(“charged tRNA”)

Accurate translation requires 2 steps:

1. a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase

2. a correct match between the tRNA anticodon & mRNA codon

Active site binds theamino acid and ATP.

Appropriate tRNA covalently bonds to amino acid, displacing AMP.

Ribosomes• Ribosomes facilitate specific coupling of

tRNA anticodons with mRNA codons in protein synthesis

• The two ribosomal subunits (large and small) are made of proteins & ribosomal RNA (rRNA)

tRNAmolecules

Exit tunnelGrowingpolypeptide

Largesubunit

mRNA 3

Computer model of functioning ribosome

Smallsubunit

5

similar in prokaryotes & eukaryotes, but differences are medically significant

note:tetracycline & streptomycin inactivate prokaryotic ribosomes

• A ribosome has three binding sites for tRNA:

1. the A site holds the tRNA that carries the next amino acid to be added to the chain

2. the P site holds the tRNA that carries the growing polypeptide chain

3. the E site is the exit site, where discharged tRNAs leave the ribosome

Exit tunnel

A site (Aminoacyl-tRNA binding site)

Smallsubunit

Largesubunit

P A

P site (Peptidyl-tRNAbinding site)

mRNAbinding site

(b) Schematic model showing binding sites

E site (Exit site)

E

Amino end

mRNA

E

5 Codons

3

tRNA

Growing polypeptide

Next aminoacid to beadded topolypeptidechain

A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

Building a Polypeptide

• The three stages of translation:

1. Initiation

2. Elongation

3. Termination

– All three stages require protein “factors” that aid in the process

Ribosome Association & Initiation of TranslationThe initiation stage of translation brings together:

1. mRNA

2. a tRNA with the first amino acid

3. the two ribosomal subunits

Met

GTPInitiator tRNA

mRNA

53

mRNA binding site

Smallribosomalsubunit

Start codon

P site

5 3

Translation initiation complex

E A

Large ribosomalsubunit

GDP

Met

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

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

Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site

the A site is available to the tRNA bearing the next amino acid

Amino endof polypeptide

mRNAE

Psite

Asite

3

5

Amino endof polypeptide

mRNAE

Psite

Asite

GTP

GDP

E

P A

3

5

Amino endof polypeptide

mRNAE

Psite

Asite

GTP

GDP

E

P A

E

P A

3

5

Amino endof polypeptide

mRNAE

Psite

Asite

GTP

GDP

E

P A

E

P A

GDPGTP

Ribosome ready fornext aminoacyl tRNA

E

P A

3

5

Elongation amino acids are

added 1 by 1 to the preceding amino acid

Each addition involves proteins called elongation factors

Codon recognition

1

Peptide bond formation

2

Translocation

3

Termination of Translation

• Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

• The A site accepts a protein called a release factor

– causes the addition of a water molecule instead of an amino acid

• This reaction releases the polypeptide, and the translation assembly then comes apart

3

The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.

The two ribosomal subunitsand the other componentsof the assembly dissociate.

Releasefactor

Stop codon(UAG, UAA, or UGA)

5

3

5

3

5

Freepolypeptide

When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.

Polyribosomes• A number of ribosomes can translate a single mRNA simultaneously, forming a

polyribosome

(or polysome)

• Polyribosomes enable a cell to make many copies of a polypeptide very quickly

Ribosomes

mRNA

0.1 mThis micrograph shows a large polyribosome in a prokaryotic cell (TEM).

An mRNA molecule is generally translated simultaneouslyby several ribosomes in clusters called polyribosomes.

Incomingribosomalsubunits

Growingpolypeptides

End ofmRNA(3 end)

Start ofmRNA(5 end)

Polyribosome

Completedpolypeptides

Functional Protein

• During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape

• Often translation is not sufficient to make a functional protein– Polypeptide chains are modified after translation

• Proteins may require post-translational modifications– Some polypeptides are activated by enzymes that cleave them– Other polypeptides come together to form the subunits of a protein

• Completed proteins are targeted to specific sites in the cell

Ribosomes

• Two populations of ribosomes are evident in cells: – free ribsomes (in the cytosol)

– bound ribosomes (attached to the ER)

• Free ribosomes mostly synthesize proteins that function in the cytosol

• Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell

• Ribosomes are identical and can switch from free to bound

• Polypeptide synthesis always begins in the cytosol

• Synthesis finishes 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 and its ribosome to the ER

Ribosome

mRNA

Signalpeptide

SRP

1

SRPreceptorprotein

Translocationcomplex

ERLUMEN

2

3

45

6

Signalpeptideremoved

CYTOSOL

Protein

ERmembrane

• Mutations-are changes in the genetic material of a cell or virus

– Spontaneous mutations can occur during DNA replication, recombination, or repair

– Mutagens are physical or chemical agents that can cause mutations

• Point mutations-are chemical changes in just one base pair of a gene

– can lead to the production

of an abnormal protein

Divided into 2 general categories:

1. Nucleotide-pair substitutions

2. One or more nucleotide-pair insertions or deletions

• Silent mutations-

have no effect on the amino acid produced by a codon because of redundancy in the genetic code

• Missense mutations-

still code for an amino acid, but not necessarily the right amino acid

• Nonsense mutations-

change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein

replaces one nucleotide and its partner with another pair of nucleotides

more

common

Nucleotide-pair substitution

Insertions and deletions-

are additions or losses of nucleotide pairs in a gene

These mutations have a disastrous effect on the resulting protein more often than substitutions do

Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation

Nucleotide-pair insertion/ deletion

prokaryotes vs. eukaryotes• Prokaryotic cells lack a nuclear envelope,

allowing translation to begin while transcription progresses

• In a eukaryotic cell:– The nuclear envelope separates transcription from translation– Extensive RNA processing occurs in the nucleus

What Is a Gene? Revisiting the Question

• The idea of the gene itself is a unifying concept of life

• 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 genegene 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