chapter 17 from gene to protein · 2018. 9. 6. · products of gene expression one gene –one...
TRANSCRIPT
Chapter 17
From Gene to
Protein
The Flow of Genetic Information
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 the links between genotype and phenotype
Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation
Central Dogma of Biology
RNA
DNA
Protein
Transcription
Translation
Replication
Concept 17.1Genes specify proteins via
transcription and translation
• The process of producing an mRNA transcript from a DNA template is known as transcription• Facilitated by RNA polymerase
• The process of producing polypeptides from mRNA is known as translation• Facilitated by ribosomes
• To determine the sequence of amino acids based on the DNA sequence, the genetic code can be used to interpret the DNA sequence
Archibald Garrod
Studied inherited metabolic conditions.
First to suggest that genes dictate phenotypes
through enzymes that catalyze specific chemical
reactions in the cell.
E.g. alkaptonuria
Symptom of black urine caused by alkapton
Garrod suggested that lack of the enzyme which
metabolizes alkapton is the inherited trait
Links gene to protein to phenotype
George Beadle and Edward Tatum
Worked with the bread mold Neurospora and
created mutants with x-rays
Mutants could not survive on the minimal medium
that was sufficient to support growth of the wild-
type Neurospora
Beadle and Tatum then hypothesized that the
mutants lacked the ability to synthesize certain
necessary molecules
George Beadle and Edward Tatum
To determine the individual specific defects for
each mutant, they selected for mutants by
including one nutrient at a time
This allowed them to identify mutants based on the
nutrient it could not synthesize
They hypothesized that these mutants lacked a
necessary enzyme for the synthesis of that nutrient
One gene – one enzyme hypothesis
The function of a gene is to dictate the production of
a specific enzyme
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
Minimal
medium
(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 Ornithine
Enzyme B Enzyme B Enzyme B
Citrulline Citrulline Citrulline Citrulline
Enzyme C Enzyme C Enzyme C Enzyme C
Arginine Arginine Arginine Arginine
Products of Gene Expression
One gene – one enzyme was a valid hypothesis at
the time.
But, not all proteins are enzymes. The phrase was
then modified to one gene – one protein.
However, proteins can be composed of multiple
polypeptides. The phrase was then again restated
as one gene – one polypeptide.
The processes involved with going from gene to
protein are known as transcription and translation
Transcription
Genes provide the instructions for protein synthesis
in the form of a nucleotide sequence.
This sequence is used to generate a single stranded
RNA molecule through the process of transcription
RNA differs from DNA in the following ways:
Single-stranded instead of double-stranded
Ribose instead of deoxyribose
Uracil (U) instead of thymine (T)
Transcription
This RNA is known as messenger RNA (mRNA).
In eukaryotes, the mRNA that is produced contains
some information that is not included in the final
polypeptide.
This initial mRNA known as pre-mRNA must undergo
RNA processing to form a mature mRNA that
contains only regions that code for a polypeptide
sequence.
This pre-mRNA is also known as a primary transcript
Translation
After the production of the mRNA from the gene
through transcription, a polypetide can be
synthesized from the mRNA through the process of
translation.
The instructions held in the sequence of the mRNA
direct the formation of a polypeptide by ribosomes,
which work to link amino acids together in the
correct order to form a polypeptide chain.
These instructions are given in the sequence of the
nucleotide bases which can be deciphered using
the genetic code.
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
(a) Bacterial cell
Nuclearenvelope
TRANSCRIPTION
RNA PROCESSINGPre-mRNA
DNA
mRNA
TRANSLATION Ribosome
Polypeptide
(b) Eukaryotic cell
The Genetic Code
There are 4 nucleotide bases that make up DNA
sequences.
However, there are 20 amino acids for which they
provide the code.
This is possible through a triplet code. Each possible
combination of three nucleotide bases codes for an
amino acid.
The instructions for the sequence of amino acids in
a polypeptide are written in non-overlapping three-
nucleotide words.
Codons
During transcription, one of the two DNA strands called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript
The template strand is read in the 3’ to 5’ direction
During translation, the mRNA base triplets, called codons, are read by translation machinery in the 5to 3 direction
Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide
Each codon specifies the addition of one of 20 amino acids
DNAmolecule
Gene 1
Gene 2
Gene 3
DNAtemplatestrand
TRANSCRIPTION
TRANSLATION
mRNA
Protein
Codon
Amino acid
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” signals to end translation
The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid
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 highly conserved. Most
organisms from the simplest to the most complex
use the same code
Gene sequences from one species can be
transcribed and translated in another species when
transferred.
Examples:
tobacco plant expressing a firefly gene
Insulin production by yeast or E. coli
Concept 17.2Transcription is the DNA-directed
synthesis of RNA: a closer look
• Transcription involves three phases: initiation,
elongation, and termination.
• The initial transcript that is produced is the pre-
mRNA which needs RNA processing to produce
the final mature mRNA which will be used for
translation
Synthesis of an RNA Transcript
As with DNA replication, there are many enzymes involved with the synthesis of mRNA during transcription.
RNA polymerase produces the new mRNA strand from the template strand of the DNA
However, since RNA is being formed, uracil is used instead of thymine
There are three stages of transcription:
Initiation
Elongation
Termination
Initiation of Transcription
The stretch of DNA that is transcribed is called a transcription unit
The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator
Transcription factors mediate the binding of RNA polymerase and the initiation of transcription
The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex
A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes
Promoter Transcription unit
Start pointDNA
RNA polymerase
5533
Initiation1
2
3
5533
Unwound
DNA
RNAtranscript
Template strand
of DNA
Elongation
Rewound
DNA
5
55
5
5
33
3
3
RNA
transcriptTermination
5533
35Completed RNA transcript
Newly made
RNA
Template
strand of DNA
Direction oftranscription(“downstream”)
3 end
RNA
polymerase
RNA nucleotides
Nontemplate
strand of DNAElongation
Elongation of the RNA transcript
As RNA polymerase moves along the DNA, it
untwists the double helix, 10 to 20 bases at a time
Transcription progresses at a rate of 40 nucleotides
per second in eukaryotes
A gene can be transcribed simultaneously by
several RNA polymerases
Termination of Transcription
The mechanisms of termination are different in
bacteria and eukaryotes
In bacteria, the polymerase stops transcription at
the end of the terminator
In eukaryotes, the polymerase continues
transcription after the pre-mRNA is cleaved from the
growing RNA chain; the polymerase eventually falls
off the DNA
Concept 17.3Eukaryotic cells modify RNA after
transcription
• RNA processing of pre-RNA involves RNA splicing
which is the removal of introns and joining of exons of the primary transcript by spliceosomes
• The ends of the pre-RNA are also modified with a
5’ cap and a 3’ poly-A tail
Ends of the mRNA strand
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
These modifications share several functions: They seem to facilitate the export of mRNA
They protect mRNA from hydrolytic enzymes
They help ribosomes attach to the 5 end
Protein-coding segmentPolyadenylation signal3
3 UTR5 UTR
5
5 Cap Start codon Stop codon Poly-A tail
G P PP AAUAAA AAA AAA…
RNA splicing
Most eukaryotic genes and their RNA transcripts
have long noncoding stretches of nucleotides that
lie between coding regions
These noncoding regions are called intervening
sequences, or introns
The other regions are called exons because they
are eventually expressed, usually translated into
amino acid sequences
RNA splicing removes introns and joins exons,
creating an mRNA molecule with a continuous
coding sequence
Pre-mRNA
mRNA
Codingsegment
Introns cut out andexons spliced together
5 Cap
Exon Intron5
1 30 31 104
Exon Intron
105
Exon
146
3
Poly-A tail
Poly-A tail5 Cap
5 UTR 3 UTR1 146
In some cases, RNA splicing is carried out by
spliceosomes
Spliceosomes consist of a variety of proteins and
several small nuclear ribonucleoproteins (snRNPs)
that recognize the splice sites
RNA splicing
RNA transcript (pre-mRNA)
Exon 1 Exon 2Intron
Protein
snRNA
snRNPs
Otherproteins
5
5
Spliceosome
Spliceosomecomponents
Cut-outintron
mRNA
Exon 1 Exon 25
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
Three properties of RNA enable it to function as an
enzyme
It can form a three-dimensional structure because of
its ability to base pair with itself
Some bases in RNA contain functional groups
RNA may hydrogen-bond with other nucleic acid
molecules
Alternative RNA Splicing
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 regions called domains
In many cases, different exons code for the different
domains in a protein
Exon shuffling may result in the evolution of new proteins
Gene
DNA
Exon 1 Exon 2 Exon 3Intron Intron
Transcription
RNA processing
Translation
Domain 2
Domain 3
Domain 1
Polypeptide
Concept 17.4Translation is the RNA-directed
synthesis of a polypeptide: a closer
look
• Translation is facilitated by ribosomes and tRNAs
• tRNAs have an anticodon that recognizes the complementary codon of the mRNA sequence
and directs the addition of amino acids
Molecular Components of
Translation
A cell translates an mRNA message into protein with
the help of transfer RNA (tRNA)
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 complementary codon
on mRNA
Polypeptide
Ribosome
Aminoacids
tRNA withamino acidattached
tRNA
Anticodon
Codons 35
mRNA
The Structure and Function of
Transfer RNA (tRNA)
A tRNA molecule consists of a single RNA strand that
is only about 80 nucleotides long
When flattened into one plane, a tRNA molecule
looks like a cloverleaf due to base pairing in regions
of the molecule
However, due to hydrogen bonds, tRNA actually
twists and folds into a three-dimensional molecule
that forms a shape that is more similar to an capital
letter L
The bottom loop is where the anticodon is located
Amino acidattachment site
(a) Two-dimensional structure
Hydrogenbonds
Anticodon
3
5
Amino acidattachment site
3
3
5
5
Hydrogenbonds
Anticodon Anticodon
(b) Three-dimensional structure(c) Symbol used
in this book
Translation
Accurate translation requires two 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 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
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”)
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)
Binding Sites
A ribosome has three binding sites for
tRNA: The E site is the exit site, where discharged tRNAs leave
the ribosome
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
Growingpolypeptide Exit tunnel
Largesubunit
Smallsubunit
tRNAmolecules
E P A
mRNA5 3
(a) Computer model of functioning ribosome
P site (Peptidyl-tRNAbinding site)
E site(Exit site)
A site (Aminoacyl-tRNA binding site)
E P A Largesubunit
mRNAbinding site
Smallsubunit
(b) Schematic model showing binding sites
Amino end Growing polypeptide
Next amino acidto be added topolypeptide chain
mRNAtRNAE
3
5 Codons
(c) Schematic model with mRNA and tRNA
Building a Polypeptide
The three stages of translation:
Initiation
Elongation
Termination
All three stages require protein “factors” that aid in
the translation process
Initiation of Translation
The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits
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 that completes the translation initiation complex
3
35
5U
U
AA
C
G
GTP GDPInitiator
tRNA
mRNA
53
Start codon
mRNA binding site
Smallribosomalsubunit
5
P site
Translation initiation complex
3
E A
Largeribosomalsubunit
Elongation of a Polypeptide Chain
During the elongation stage, amino acids are
added one by one to the preceding amino acid
Each addition involves proteins called elongation
factors and occurs in three steps: codon
recognition, peptide bond formation, and
translocation
Amino endof polypeptide
mRNA
5
3E
Psite
Asite
GTP
GDP
E
P A
E
P A
GDP
GTP
Ribosome ready fornext aminoacyl tRNA
E
P A
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
Releasefactor
3
5
Stop codon(UAG, UAA, or UGA)
5
3
2
Freepolypeptide
2 GDP
GTP
5
3
Ribosomes
mRNA
(b) 0.1 µm
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
Growingpolypeptides
Completedpolypeptide
Incomingribosomalsubunits
Start ofmRNA(5 end)
End ofmRNA(3 end)(a)
Post-Translational Modifications
Often translation is not sufficient to make a functional protein
Polypeptide chains are modified after translation
Completed proteins are targeted to specific sites in the cell
During and after synthesis, a polypeptide chain spontaneously coils and folds into its three-dimensional shape
Proteins may also require post-translational modifications before doing their job
Some polypeptides are activated by enzymes that cleave them
Other polypeptides come together to form the subunits of a protein
Ribosome Location
Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and 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
Targeting Polypeptides
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
Signal-recognitionparticle (SRP)
CYTOSOLTranslocationcomplex
SRPreceptorprotein
ER LUMEN
Signalpeptideremoved
ERmembrane
Protein
Concept 17.5Point mutations can affect protein
structure and function
• Mutations change DNA sequences
• A mutation in the coding region of DNA can change the resulting protein through changes in in amino acids
• Different mutations can have differences in severity in terms of the effect on the resulting protein
Mutagens
Mutations result in changes in the original DNA
sequence
Spontaneous mutations can occur during DNA
replication, recombination, or repair
Mutagens are physical or chemical agents that can
cause mutations
Point mutation – Substitution
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 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
Point Mutation – 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
Wild-type
3DNA template strand
5
5
5
3
3
Stop
Carboxyl endAmino end
Protein
mRNA
3
3
3
5
5
5
A instead of G
U instead of C
Silent (no effect on amino acid sequence)
Stop
T instead of C
3
3
3
5
5
5
A instead of G
Stop
Missense
A instead of T
U instead of A
3
3
3
5
5
5
Stop
Nonsense No frameshift, but one amino acid missing (3 base-pair deletion)
Frameshift causing extensive missense (1 base-pair deletion)
Frameshift causing immediate nonsense (1 base-pair insertion)
5
5
53
3
3
Stop
missing
missing
3
3
3
5
5
5
missing
missing
Stop
5
5
53
3
3
Extra U
Extra A
(a) Base-pair substitution (b) Base-pair insertion or deletion
Concept 17.6While gene expression differs
among the domains of life, the
concept of a gene is universal
• Gene expression differs between the different
domains of life but the general concept remains consistent
• DNA carries genetic information in the form of
genes, which can be transcribed and translated
to produce a final product in the form of a
polypeptide or a protein
Gene expression in different
domains
Differences in mechanics of gene expression between bacteria and eukarya: Different RNA polymerases
Different methods for termination of transcription
Different ribosomes
Archaea tend to be more similar to eukarya for these categories
Bacteria can simultaneously transcribe and translate the same gene
In eukarya, transcription and translation are separated by the nuclear envelope
In archaea, transcription and translation are likely coupled
RNA polymerase
DNA
Polyribosome
mRNA
0.25 µmDirection oftranscription
DNA
RNApolymerase
Polyribosome
Polypeptide(amino end)
Ribosome
mRNA (5 end)
Genes
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
TRANSCRIPTION
RNA PROCESSING
DNA
RNAtranscript
3
5RNApolymerase
RNA transcript(pre-mRNA)
Intron
Exon
NUCLEUS
Aminoacyl-tRNAsynthetase
AMINO ACID ACTIVATION
Aminoacid
tRNACYTOPLASM
Growingpolypeptide
3
Activatedamino acid
mRNA
TRANSLATION
Ribosomalsubunits
5
E
P
A
AAnticodon
Ribosome
Codon
E
Sense and Antisense
3’ TACATCGCCCATAACGAGAAT 5’
5’ 3’
Template Strand
(Antisense)
Coding Strand
(Sense)
mRNA5’ 3’
polypeptide