From DNA to Protein: Genotype to Phenotype

12From DNA to Protein: Genotype to Phenotype

12 From DNA to Protein: Genotype to Phenotype• Review

12.1 What Is the Evidence that Genes Code for Proteins?

12.2 How Does Information Flow from Genes to Proteins?

• 12.3 How Is the Information Content in DNA Transcribed to Produce RNA?

• 12.4 How Is RNA Translated into Proteins?

• 12.5 What Happens to Polypeptides after Translation?

• 12.6 What Are Mutations?

12-1 Recap:• Beadle & Tatum’s studies of Beadle & Tatum’s studies of

mutations in bread molds led to mutations in bread molds led to the the one gene-one polypeptide one gene-one polypeptide hypothesishypothesis::

• The function of a gene is to code The function of a gene is to code for a specific polypeptidefor a specific polypeptide

12-1 Recap ?s:

• What’s a “model organism”?What’s a “model organism”?

• Why was Why was NeurosporaNeurospora a good model for a good model for studying biochemical genetics?studying biochemical genetics?

• How were Beadle and Tatum’s expts set How were Beadle and Tatum’s expts set up to determine, on the basis of up to determine, on the basis of phenotypes of mutant strains, the order phenotypes of mutant strains, the order of a biochemical pathway?of a biochemical pathway?

• What’s the distinction between What’s the distinction between “protein” and “polypeptide”?“protein” and “polypeptide”?


The molecular basis of phenotypes was known before it was known that DNA is the genetic material.

Studies of many different organisms showed that major phenotypic differences were due to specific proteins.


Model organisms:

easy to grow or observe; show the phenomenon to be studied

results from one organism applied to others

Examples: pea plants, Drosophila, E. coli, common bread mold Neurospora crassa


Bread mold experiments:

Suggested the

one-gene, one-enzyme hypothesis

Figure 12.1 One Gene, One Enzyme (Part 1)

Figure 12.1 One Gene, One Enzyme (Part 2)

each mutant was missing a single enzyme in the pathway.


The gene-enzyme relationship has been revised to the one-gene, one-polypeptide relationship.

Example: In hemoglobin, each polypeptide chain is specified by a separate gene.

Other genes code for RNA that is not translated to polypeptides; some genes are involved in controlling other genes.

12-2 Recap:• Central Dogma of molec bio:Central Dogma of molec bio:

DNA codes for the production of RNA, RNA DNA codes for the production of RNA, RNA codes for the production of polypeptides codes for the production of polypeptides (proteins)(proteins)

• Proteins do NOT code for the production of Proteins do NOT code for the production of protein, DNA, or RNAprotein, DNA, or RNA

• Transcription = process that copies a DNA Transcription = process that copies a DNA sequence into mRNAsequence into mRNA

• Translation = process by which this info is Translation = process by which this info is converted into proteinconverted into protein

• tRNA recognizes the genetic info in mRNA and tRNA recognizes the genetic info in mRNA and brings the appropriate amino acid into brings the appropriate amino acid into position in a growing polypeptide chainposition in a growing polypeptide chain

12-2 Recap ?s:

• What’s that central dogma all What’s that central dogma all about?about?

• What are the roles of mRNA and What are the roles of mRNA and tRNA in gene expression?tRNA in gene expression?


Expression of a gene to form a polypeptide:

• Transcription—copies information from gene to a sequence of RNA.

• Translation—converts RNA sequence to amino acid sequence.


RNA, ribonucleic acid differs from DNA:

• Usually one strand

• The sugar is ribose

• Contains uracil (U) instead of thymine (T)


RNA can pair with a single strand of DNA, except that adenine pairs with uracil instead of thymine.

RNA (CODONS)

Practice! DNA RNA

Figure 12.2 The Central Dogma

The central dogma of molecular biology: information flows in one direction when genes

are expressed (Francis Crick).


The central dogma raised two questions:

• How does genetic information get from the nucleus to the cytoplasm?

• What is the relationship between a DNA sequence and an amino acid sequence?


One hypothesis—

messenger RNA (mRNA) forms as a complementary copy of DNA and carries information to the cytoplasm.

This process is transcription.

Figure 12.3 From Gene to Protein


Other hypothesis—an adapter molecule that can bind amino acids, and recognize a nucleotide sequence—transfer RNA (tRNA).

tRNA molecules carrying amino acids line up on mRNA in proper sequence for the polypeptide chain—translation.


Exception to the central dogma:

Viruses: acellular particles that reproduce inside cells; many have RNA instead of DNA.


Synthesis of DNA from RNA is reverse transcription.

Viruses that do this are retroviruses.

12-3 Recap:• Transcription (catalyzed by an Transcription (catalyzed by an

RNA polymerase) proceeds in 3 RNA polymerase) proceeds in 3 steps:steps:

• Initiation, elongation, terminationInitiation, elongation, termination

• Genetic code relates the Genetic code relates the information in mRNA (linear information in mRNA (linear sequence of codons) to protein sequence of codons) to protein (linear sequence of amino acids)(linear sequence of amino acids)

12-3 Recap ?s:

• What are the steps of gene What are the steps of gene transcription (producing mRNA)?transcription (producing mRNA)?

• How do RNA polymerases work?How do RNA polymerases work?

• How was the genetic code How was the genetic code deciphered?deciphered?

12.3 How Is the Information Content in DNA Transcribed to Produce RNA?

Within each gene, only one strand of DNA is transcribed—the template strand.

Transcription produces mRNA;

the same process is used to produce tRNA and rRNA.


RNA polymerases catalyze synthesis of RNA.

single enzyme-template binding results in polymerization of hundreds of RNA bases.

Figure 12.4 RNA Polymerase


Transcription occurs in three phases:

• Initiation

• Elongation

• Termination


Initiation requires a promoter—a special sequence of DNA.

RNA polymerase binds to it.

tells RNA polymerase where to start, which direction to go in, and which strand of DNA to transcribe.

Part of it is the initiation site.

Figure 12.5 DNA Is Transcribed to Form RNA

RNA polymerase binds to the promoter and starts to unwind the DNA strands.

Templatestrand

Promoter Unwinding of DNA

Rewinding of DNA

Complementary strand

RNA polymerase

Initiation site Termination site

3′

5′

3′

5′

5′


3′

5′

3′

5′

3′

5′

Direction of transcription

RNA polymerase reads the DNA template strand from 3′ to 5′ and produces the RNA transcript by adding nucleotides to the 3′ end.

3′

5′

3′

5′

5′

3′

RNA transcript

Nucleoside triphosphates(A, U, C, G)


3′

5′

3′

5′

3′ 5′

RNA

When RNA polymerase reaches the termination site, the RNA transcript is set free from the template.


Elongation: RNA polymerase unwinds DNA about 10 base pairs at a time; reads template in 3′ to 5′ direction.

The RNA transcript is antiparallel to the DNA template strand.

RNA polymerases do not proofread and correct mistakes.

Figure 12.5 DNA Is Transcribed to Form RNA (B)


Termination: specified by a specific DNA sequence.

Mechanisms of termination are complex and varied.

Eukaryotes—first product is a pre-mRNA that is longer than the final mRNA and must undergo processing.

Figure 12.5 DNA Is Transcribed to Form RNA (C)


The genetic code: specifies which amino acids will be used to build a protein

Codon: a sequence of three mRNA bases. Each specifies a particular amino acid.

Start codon: AUG—initiation signal for translation

Stop codons: stops translation and polypeptide is released

Figure 12.6 The Genetic Code

AUG UAC CAU UUA GCC AUC AAC UUU UAC UAU AAU UGA

ANIMATIONS!

• Transcription

• Deciphering the Genetic Code


For most amino acids, there is more than one codon; the genetic code is redundant.

But not ambiguous— each codon specifies only one amino acid.


The genetic code is nearly universal: codons that specify amino acids are the same in all organisms!!

Exceptions: within mitochondria and chloroplasts, and in one group of protists.


This common genetic code is a common language for evolution.Ancient; has remained intact also facilitates genetic engineering


How was the code deciphered?

20 “code words” (amino acids) are written with only four “letters.”

Triplet code seemed likely: could account for 4 × 4 × 4 = 64 codons.

12-4 Recap:• Key step in protein synthesis: Key step in protein synthesis:

attachment of amino acid to attachment of amino acid to proper tRNA (activating enzyme)proper tRNA (activating enzyme)

• Translation of genetic info from Translation of genetic info from mRNA into protein occurs @ mRNA into protein occurs @ ribosomeribosome

• Multiple ribosomes may act on a Multiple ribosomes may act on a single mRNA to make multiple single mRNA to make multiple copies of the protein for which it copies of the protein for which it codescodes

12-4 Recap ?s:

• How is an amino acid attached to a How is an amino acid attached to a specific tRNA?specific tRNA?

• Why’s it called “second genetic Why’s it called “second genetic code”?code”?

• Describe initiation, elongation, and Describe initiation, elongation, and termination of translation.termination of translation.

12.4 How Is RNA Translated into Proteins?

tRNA: for each amino acid, there’s a specific type

Functions:

• Carries an amino acid

• Associates with mRNA molecules

• Interacts with ribosomes

Figure 12.8 Transfer RNA

Video: tRNA


The conformation (3D shape) of tRNA results from base pairing (H bonds) within the molecule.

3′ end is the amino acid attachment site—binds covalently. Always CCA.

Anticodon: site of base pairing with mRNA. Unique for each tRNA.

Figure 12.9 Charging a tRNA Molecule

tRNA site

Specific amino acid(e.g., alanine) Pyrophosphate (PPi)

Pi

Alanine-specific tRNA

The enzyme activates the amino acid, catalyzing reaction with ATP to form high energy AMP–amino acid and a pyrophosphate ion.

The enzyme then catalyzes a reaction of the activated amino acid with the correct tRNA.

The specificity of the enzyme ensures that the correct amino acid and tRNA have been brought together.

The charged tRNA will deliver the appropriate amino acid to join theelongating polypeptide productof translation.

Activatedalanine

tRNA bonded to alanine

Activatingenzyme (aminoacyl-tRNA synthase) for a specific amino acid

Alanine

Charged tRNA

tRNA

Activating enzyme

ATP site

Amino acid site

Figure 12.9 Charging a tRNA Molecule (Part 2)


Amino acid is attached to the 3′ end of tRNA by an energy-rich bond—

provides energy for synthesis of peptide bond to join amino acids.


Ribosome: holds mRNA and tRNA in correct positions to allow assembly of polypeptide chain.

are not specific, they can make any type of protein.


Ribosomes have two subunits:

large and small

In eukaryotes, the large subunit has three molecules of ribosomal RNA (rRNA) and 45 different proteins in a precise pattern.

The small subunit has one rRNA and 33 proteins.


Subunits are held together by ionic and hydrophobic forces (not covalent bonds).

When not active in translation, the subunits exist separately.

Figure 12.10 Ribosome Structure


Large subunit has three tRNA binding sites:

• A site binds with anticodon of charged tRNA.

• P site is where tRNA adds its amino acid to the growing chain.

• E site is where tRNA sits before being released.


H bonds form between anticodon of tRNA and codon of mRNA.

Small subunit rRNA validates the match—if hydrogen bonds have not formed between all three base pairs, it must be an incorrect match, and the tRNA is rejected.


Translation also occurs in three steps:

• Initiation

• Elongation

• Termination


Initiation:

An initiation complex forms—

charged tRNA and small ribosomal subunit, both bound to mRNA.

rRNA binds to recognition site on mRNA, “upstream” from the start codon.

Figure 12.11 The Initiation of Translation (Part 1)

Figure 12.11 The Initiation of Translation (Part 2)


Start codon is AUG; first amino acid is always methionine, which may be removed after translation.

The large subunit joins the complex, the charged tRNA is now in the P site of the large subunit.

Initiation factors are responsible for assembly of the initiation complex.


Elongation: 2nd charged tRNA enters A site.

Large subunit catalyzes two reactions:

• Breaks bond between tRNA in P site and its amino acid.

• Peptide bond forms between that amino acid and amino acid on tRNA in A site.

Figure 12.12 The Elongation of Translation (Part 1)

Figure 12.12 The Elongation of Translation (Part 2)


1st tRNA releases its methionine,

moves to E site

dissociates from ribosome—can then become charged again.

Elongation occurs as the steps are repeated, assisted by proteins called elongation factors.


Termination: translation ends when a stop codon enters the A site.

binds a protein release factor—allows hydrolysis of bond between polypeptide chain and tRNA on P site.

Polypeptide chain—C terminus is last amino acid added.

Figure 12.13 The Termination of Translation (Part 1)



Table 12.1

Last Animation!

• Protein Synthesis


Several ribosomes can work together to translate the same mRNA, producing multiple copies of the polypeptide.

A strand of mRNA with associated ribosomes is called a polyribosome or polysome.

Figure 12.14 A Polysome (Part 1)

Figure 12.14 A Polysome (Part 2)

12-5 Recap:

12-5 Recap ?s:

12.5 What Happens to Polypeptides after Translation?

Posttranslational aspects of protein synthesis:

Polypeptide may be moved from synthesis site to an organelle, or out of the cell.

Polypeptides are often modified with more chemical groups.


Polypeptide folds as it emerges from the ribosome.

The amino acid sequence determines the pattern of folding.

Amino acid sequence also contains a signal sequence—an “address label.”


Amino acid sequence gives a set of instructions:

“Finish translation and send to an organelle.”

OR

“Stop translation, go to the ER, finish synthesis there.”

Figure 12.15 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell


If the protein is sent to the ER:

• Signal sequence binds to a signal receptor particle, before translation is done.

• Ribosome attaches to a receptor on the ER, the growing polypeptide chain passes through the channel.

• An enzyme removes the signal sequence.

Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER

Rough endoplasmic reticulum

Inside of cell

Interior of RER

Receptor protein

3′

5′

Ribosome

Protein synthesis begins on free ribosomes in the cytosol. The signal sequence is at the N-terminal end of the polypeptide chain.

1Signal sequence

Signal recognitionparticle

ERmembrane

mRNA

3′

5′



2 The polypeptide binds to a signal recognition particle, and both bind to a receptor protein in the membrane of the ER.



3 The signal recognition particle is released. The signal sequence passes through a channel in the receptor.

3′

Enzyme for removal



3′

The signal sequence is removed by an enzyme in the ER.

4



3′

The polypeptide continues to elongate.

5



3′

Translation terminates.6



The ribosome is released. The protein folds inside the ER.

7


Sugars may be added in Golgi apparatus—

the resulting glycoproteins end up in the plasma membrane, lysosomes, or vacuoles.


Protein modifications:

Proteolysis: cutting the polypeptide chain, by proteases.

Glycosylation: addition of sugars to form glycoproteins.

Phosphorylation: addition of phosphate groups by kinases. Charged phosphate groups change the conformation.

Figure 12.17 Posttranslational Modifications of Proteins

12-6 Recap:

12-6 Recap ?s:

12.6 What Are Mutations?

Somatic mutations occur in somatic (body) cells. Mutation is passed to daughter cells, but not to sexually produced offspring.

Germ line mutations occur in cells that produce gametes. Can be passed to next generation.


Conditional mutants: express phenotype only under restrictive conditions.

Example: the allele may code for an enzyme that is unstable at certain temperatures.


All mutations are alterations of the nucleotide sequence.

Point mutations: change in a single base pair—loss, gain, or substitution of a base.

Chromosomal mutations: change in segments of DNA—loss, duplication, or rearrangement.


Point mutations can result from replication and proofreading errors, or from environmental mutagens.

Silent mutations have no effect on the protein because of the redundancy of the genetic code.

Silent mutations result in genetic diversity not expressed as phenotype differences.



Missense mutations: base substitution results in amino acid substitution.


Sickle allele for human β-globin is a missense mutation.

Sickle allele differs from normal by only one base—the polypeptide differs by only one amino acid.

Individuals that are homozygous have sickle-cell disease.

Figure 12.18 Sickled and Normal Red Blood Cells

Photo 12.1 Normal human erythrocytes. LM, differential interference contrast.

Photo 12.2 Sickled human erythrocytes (sickle-cell anemia). LM, differential interference contrast.

Photo 12.3 Computer-simulated space-filling model of phenylalanine transfer RNA (tRNA).


Nonsense mutations: base substitution results in a stop codon.


Frame-shift mutations: single bases inserted or deleted—usually leads to nonfunctional proteins.


Chromosomal mutations:

Deletions—severe consequences unless it affects unnecessary genes or is masked by normal alleles.

Duplications—if homologous chromosomes break in different places and recombine with the wrong partners.

Figure 12.19 Chromosomal Mutations (A, B)


Chromosomal mutations:

Inversions—breaking and rejoining, but segment is “flipped.”

Translocations—segment of DNA breaks off and is inserted into another chromosome. Can cause duplications and deletions. Meiosis can be prevented if chromosome pairing is impossible.

Figure 12.19 Chromosomal Mutations (C, D)


Spontaneous mutations—occur with no outside influence. Several mechanisms:

• Bases can form tautomers—different forms; rare tautomer can pair with the wrong base.

• Chemical reactions may change bases (e.g., loss of amino group).


• Replication errors—some escape detection and repair.

• Nondisjunction in meiosis.


Induced mutation—due to an outside agent, a mutagen.

Chemicals can alter bases (e.g., nitrous acid can cause deamination).

Some chemicals add other groups to bases (e.g., benzpyrene adds a group to guanine and prevents base pairing). DNA polymerase will then add any base there.


Ionizing radiation such as X-rays create free radicals—highly reactive—can change bases, break sugar phosphate bonds.

UV radiation is absorbed by thymine, causing it to form covalent bonds with adjacent nucleotides—disrupts DNA replication.

Figure 12.20 Spontaneous and Induced Mutations (Part 1)

Figure 12.20 Spontaneous and Induced Mutations (Part 2)


Mutation provides the raw material for evolution in the form of genetic diversity.

Mutations can harm the organism, or be neutral.

Occasionally, a mutation can improve an organism’s adaptation to its environment, or become favorable as conditions change.


Complex organisms tend to have more genes than simple organisms.

If whole genes are duplicated, the new genes would be surplus genetic information.

Extra copies could lead to the production of new proteins.

New genes can also arise from transposable elements (see Chapters 13 and 14).

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From DNA to Protein: Genotype to Phenotype