From Genes to ProteinsChapter 17
AP Chapter 17
How was the fundamental relationship between genes and proteins discovered?
In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions
He studied alkaptonuria.
• Alkaptonuria is best known for the darkening of urine from yellow to brown to black after it is exposed to the air.
• Garrod studied the patterns in several families, realized it followed an autosomal recessive pattern of inheritance, and postulated that it was caused by a mutation in a gene encoding an enzyme involved in the metabolism of a class of compounds called alkaptans.
Nutritional Mutants in Neurospora: Scientific Inquiry
• 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.
• They identified mutants that lacked certain enzymes necessary for synthesizing.
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-2
RESULTS
EXPERIMENT
CONCLUSION
Growth:Wild-typecells growing and dividing
No growth:Mutant cellscannot grow and divide
Minimal medium
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)
Class I mutants(mutation in
gene A)Wild type
Class II mutants(mutation in
gene B)
Class III mutants(mutation in
gene C)
Gene A
Gene B
Gene C
Precursor Precursor Precursor PrecursorEnzyme A Enzyme AEnzyme A Enzyme A
Enzyme B
Ornithine Ornithine Ornithine OrnithineEnzyme B Enzyme B Enzyme B
Citrulline Citrulline Citrulline CitrullineEnzyme C Enzyme C Enzyme C Enzyme C
Arginine Arginine Arginine Arginine
The mutants couldnot produce theenzymes to movethrough the pathway.
They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme
Revision of the hypothesis…
• Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein
• Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis
Note that it is common to refer to gene products as proteins rather than polypeptides.
Remember!
Proteins are made of amino acids.
RNA is a key-player in Protein Synthesis.
• RNA differs from DNA in three major ways.
– RNA has a ribose sugar.– RNA has uracil instead of thymine.– RNA is a single-stranded structure.
URACIL
Different sugars in DNA and RNA
ribose
deoxyribose
Has oneLess oxygen
atom
Fig. 17-4
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
Basic Principles of Transcription and Translation
• Transcription is the synthesis of mRNA (messenger RNA) under the direction of DNA
• Occurs in the nucleus of eukaryotes• Translation is the synthesis of a
polypeptide, which occurs under the direction of mRNA
• Ribosomes are the sites of translation
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-3a-1
TRANSCRIPTIONDNA
mRNA
(a) Bacterial cell
Fig. 17-3a-2
(a) Bacterial cell
TRANSCRIPTIONDNA
mRNA
TRANSLATIONRibosome
Polypeptide
Differences in Euk and Prok 1. In a eukaryotic cell, the nuclear envelope
separates transcription from translation
2. Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA. A primary transcript is the initial RNA transcript from any gene.
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-3
TRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSINGPre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
The central dogma is the concept that cells are governed by a cellular chain of command: DNA RNA protein.
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
• How many bases correspond to an amino acid?
3
The Genetic Code is read in CODONS which are Triplets of Nucleotide Bases in mRNA that
code for amino acids.
• Example: AGT codes for the amino acid serine.
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Cracking the Code
• All 64 codons were deciphered by the mid-1960s by Marshall Nirenberg
• Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation
• There is one “start” code AUG which also codes for the amino acid methionine.
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-5Second mRNA base
Fir
st
mR
NA
ba
se
(5 e
nd
of
co
do
n)
Th
ird
mR
NA
ba
se
(3 e
nd
of
co
do
n)
What does this mean?
• The genetic code is redundant: an amino acid may have more than one code.
• but not ambiguous; no codon specifies more than one amino acid
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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-6
(a) Tobacco plant expressing a firefly gene
(b) Pig expressing a jellyfish gene
In a nutshell….
• During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in a mRNA.
• Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction
• Each codon specifies the addition of one of 20 amino acids
Fig. 17-4
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
DNA:TAC AAA TGA GGA TCA GCT ACC CCA ACA ACT
mRNA:
AUG UUU ACU CCU AGU CGA UGG GGU UGU UGA
Amino acids
Met(start)–phe–thr–pro–ser-arg-trp-gly-cys-stop
Transcription is the DNA-directed synthesis of RNA: a closer look
• The three stages of transcription:– Initiation– Elongation– Termination
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides.
• RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-7a-1Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
The stretch of DNA that is transcribed is called a transcription unit
The DNA sequence where RNA polymerase attaches is called the promoter.
Fig. 17-7b
Elongation
RNApolymerase
Nontemplatestrand of DNA
RNA nucleotides
3 end
Direction oftranscription(“downstream”) Template
strand of DNA
Newly madeRNA
3
5
5
Promoters (TATA box area) signal the initiation of RNA synthesis.
Transcription factors mediate the binding of RNA polymerase and the initiation of transcription
Adds at 3’of growing end
Fig. 17-8A eukaryotic promoterincludes a TATA box
3
1
2
3
Promoter
TATA box Start point
Template
TemplateDNA strand
535
Transcriptionfactors
Several transcription factors mustbind to the DNA before RNApolymerase II can do so.
5533
Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.
RNA polymerase IITranscription factors
55 53
3
RNA transcript
Transcription initiation complex
Transcription factors
Fig. 17-7a-2Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
Fig. 17-7a-3Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5
5 5 3 3 3
RNAtranscript
Fig. 17-7a-4Promoter Transcription unit
DNAStart point
RNA polymerase
553
3
Initiation
33
1
RNAtranscript
5 5
UnwoundDNA
Template strandof DNA
2 Elongation
RewoundDNA
5
5 5 3 3 3
RNAtranscript
3 Termination
5
5 5 33
3Completed RNA transcriptanimations
http://www.hhmi.org/biointeractive/dna/DNAi_transcription_vo1.html
• A gene can be transcribed simultaneously by several RNA polymerases
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Differences in transcription in Prokaryotes and Eukaryotes
1. Prok lack transcription factors2. No terminator sequence in Euk4. More types of RNA polymerases in
euk5. No RNA processing in Prok
Compare DNA polymerase and RNA polymerase
How they function:
• DNA polymerase used to bind DNA nucleotides to present DNA
• RNA polymerase used to bind RNA nucleotides to DNA
Requirement for a template and primer:
• RNA polymerase does not need a primer. Both use DNA as a template.
Direction of synthesis: 5’ to 3’
Type of nucleotides used:
DNA-A,T,C,G;
RNA – A,U,C,G (U instead of T)
What is a promoter?• It is a DNA sequence where
transcription will begin and where the RNA polymerase attaches.
• It is located upstream of a transcription unit.
What makes RNA polymerase start transcribing a gene at the right place on the DNA in a bacterial cell? In an eukaryotic cell?
• A promoter area
In an eukaryotic cell?
• A promoter area and transcription factors
Eukaryotic cells modify RNA after transcription
• Each end of a pre-mRNA molecule is modified in a particular way:–The 5 end receives a modified
nucleotide 5 cap–The 3 end gets a poly-A tail
Fig. 17-9
Protein-coding segment Polyadenylation signal3
3 UTR5 UTR
5
5 Cap Start codon Stop codon Poly-A tail
G P PP AAUAAA AAA AAA…
G
(UTR – untranslated region)
These modifications share several functions:
–They facilitate the export of mRNA out of the nucleus
–They protect mRNA from hydrolytic enzymes
–They help ribosomes attach to the 5 end
Split Genes and RNA Splicing
• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions called introns.
• Coding regions are exons
• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence
Fig. 17-10
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5 Cap
Exon Intron5
1 30 31 104
Exon Intron
105
Exon
146
3Poly-A tail
Poly-A tail5 Cap
5 UTR 3 UTR1 146
http://www.dnalc.org/resources/3d/
Remove the introns:
IJOILKJLOVEIUIOAPJKLBIOLOGYIOY
IJOILKJLOVEIUIOAPJKLBIOLOGYI
Remember the exons…..exit or go on
to make the proteins!
How are the pieces cut and spliced?
• Spliceosomes consist of a variety of proteins and small nuclear RNA (snRNA), “snRNP’s”, that recognize the splice sites
Did you call me? Oh, “snurps” not smurfs!
Fig. 17-11-1RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
Fig. 17-11-2RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
5
Spliceosome
Fig. 17-11-3RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
ProteinsnRNA
snRNPs
Otherproteins
5
5
Spliceosome
Spliceosomecomponents
Cut-outintronmRNA
Exon 1 Exon 25
• Ribozymes are catalytic RNA molecules that function as enzymes and can also splice RNA
The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins.
The Functional and Evolutionary Importance of Introns
• Alternative RNA splicing can enable some genes to encode more than one kind of protein depending on which segments are treated as exons.
• Introns allow more room for crossing-over which results in exon shuffling may result in the evolution of new proteins
A Closer Look at Translation
Fig. 17-4
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
Remember…
• Translation is using the mRNA code to order the amino acids in a polypeptide (protein).
How do the amino acids get to the ribosome?
t-RNA
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
Fig. 17-14
Amino acidattachment site
3
5
Hydrogenbonds
Anticodon
(a) Two-dimensional structure
Amino acidattachment site
5
3
Hydrogenbonds
3 5AnticodonAnticodon
(c) Symbol used in this book(b) Three-dimensional structure
Molecules of tRNA are not identical:
Each carries a specific amino acid on one end.
Each has an anticodon on the other end.
The anticodon base-pairs with a a complementary codon on mRNA
Accurate translation requires two steps:
– First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase
– Second: a correct match between the tRNA anticodon and an mRNA codon
Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon.
Fig. 17-15-1
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
Fig. 17-15-2
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
Fig. 17-15-3
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
tRNA
tRNA
Aminoacyl-tRNAsynthetase
Computer model
AMPAdenosineP
Fig. 17-15-4
Amino acid Aminoacyl-tRNAsynthetase (enzyme)
ATP
AdenosineP P P
AdenosineP
PP i
PPi
i
tRNA
tRNA
Aminoacyl-tRNAsynthetase
Computer model
AMPAdenosineP
Aminoacyl-tRNA(“charged tRNA”)
I’m really worried about my aminoacyl tRNA synthetases…
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 and ribosomal RNA (rRNA)
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• A ribosome has three binding sites for tRNA:– The P site holds the tRNA that carries the
growing polypeptide chain– The A site holds the tRNA that carries the
next amino acid to be added to the chain– The E site is the exit site, where discharged
tRNAs leave the ribosome
Remember: P first, then A, then E!
Fig. 17-16a
Growingpolypeptide Exit tunnel
tRNAmolecules
Largesubunit
Smallsubunit
(a) Computer model of functioning ribosome
mRNA
E P A
5 3
Fig. 17-16b
P site (Peptidyl-tRNAbinding site) A site (Aminoacyl-
tRNA binding site)E site(Exit site)
mRNAbinding site
Largesubunit
Smallsubunit
(b) Schematic model showing binding sites
Next amino acidto be added topolypeptide chain
Amino end Growing polypeptide
mRNAtRNA
E P A
E
Codons
(c) Schematic model with mRNA and tRNA
5
3
Fig. 17-13
Polypeptide
Ribosome
Aminoacids
tRNA withamino acidattached
tRNA
Anticodon
TrpPhe Gly
Codons 35
mRNA
Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction
Building a Polypeptide
• The three stages of translation:– Initiation– Elongation– Termination
• All three stages require protein “factors” that aid in the translation process
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Steps: Initiation1. First, a small ribosomal subunit binds
with mRNA and a special initiator tRNA
2. Then the small subunit moves along the mRNA until it reaches the start codon (AUG met) at the P site
3. Proteins called initiation factors bring in the large subunit that completes the translation initiation complex
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-17
3355U
UA
ACGMet
GTP GDPInitiator
tRNA
mRNA
5 3Start codon
mRNA binding site
Smallribosomalsubunit
5
P site
Translation initiation complex
3
E A
Met
Largeribosomalsubunit
Elongation of the Polypeptide Chain
• tRNA bearing the next amino acid comes in at the A site by codon recognition
• Amino acids are joined by peptide bonds• tRNA that was in the P site moves over into
the E site. Everything shifts over.
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
More realistic images
Fig. 17-18-1
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
Fig. 17-18-2
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
GTP
GDP
E
P A
Fig. 17-18-3
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
GTP
GDP
E
P A
E
P A
Fig. 17-18-4
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
GTP
GDP
E
P A
E
P A
GDPGTP
Ribosome ready fornext aminoacyl tRNA
E
P A
http://www.dnalc.org/resources/3d/16-translation-advanced.html
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
• The 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
Fig. 17-19-1
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
Fig. 17-19-2
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
5
32
Freepolypeptide
2 GDP
GTP
Fig. 17-19-3
Releasefactor
3
5Stop codon(UAG, UAA, or UGA)
5
32
Freepolypeptide
2 GDP
GTP
5
3H2O
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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-20
Growingpolypeptides
Completedpolypeptide
Incomingribosomalsubunits
Start ofmRNA(5 end)
Polyribosome
End ofmRNA(3 end)
(a)
Ribosomes
mRNA
(b) 0.1 µm
Protein Folding and Post-Translational Modifications
During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape
It may have sugarsand lipids bonded to it.
Targeting Polypeptides to Specific Locations
• Some will be free proteins and some will be bound 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
Let’s do this!
DNA: (in nucleus)
TAC AAA TGA GGA TCA GCT ACC CCA ACA ACT
RNA: (in cytoplasm, at ribosome)
Point mutations can affect protein structure and function
• Mutations are changes in the genetic material of a cell or virus
• Mutagens are physical or chemical agents that can cause mutations
• Point mutations are chemical changes in just one base pair of a gene
Fig. 17-22
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
33
3
3
3
3
55
5
55
5
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
Types of Point Mutations
• Point mutations within a gene can be divided into two general categories– Base-pair substitutions– Base-pair insertions or deletions
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Substitutions• A base-pair substitution replaces one
nucleotide and its partner with another pair of nucleotides
• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the 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
Insertions and Deletions
• 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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Fig. 17-23a
Wild type
3DNA templatestrand
3
355
5mRNA
Protein
Amino end
Stop
Carboxyl end
A instead of G
33
3
U instead of C
55
5
Stop
Silent (no effect on amino acid sequence)
Fig. 17-23b
Wild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
T instead of C
A instead of G
33
3
5
5
5
Stop
Missense
Fig. 17-23cWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
A instead of T
U instead of A
33
3
5
5
5
Stop
Nonsense
Fig. 17-23d
Wild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
Extra A
Extra U
33
3
5
5
5
Stop
Frameshift causing immediate nonsense (1 base-pair insertion)
Fig. 17-23e
Wild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
missing
missing
33
3
5
5
5
Frameshift causing extensive missense (1 base-pair deletion)
Fig. 17-23fWild type
DNA templatestrand
35
mRNA
Protein
5
Amino end
Stop
Carboxyl end
53
3
missing
missing
33
3
5
5
5
No frameshift, but one amino acid missing (3 base-pair deletion)
Stop
Fig. 17-22
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
33
3
3
3
3
55
5
55
5
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
NONPOLARPOLAR
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 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
Fig. 17-UN8
Top Related