Transcript Document
Chapter 17
From Gene to Protein
Overview: 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 = process by which DNA directs protein synthesis
– includes two stages: transcription and translation
Basic Principles of Transcription and Translation
• RNA is the bridge between genes and the proteins for which they
code
• Transcription = synthesis of RNA
– using information in DNA
• Translation = synthesis of a polypeptide
– using information in the mRNA
– Ribosomes - sites of translation
DNA
RNA
Protein
The Products of Gene Expression: A Developing Story
•
original hypothesis posed by scientists: one gene – one
enzyme
• BUT a lot of proteins aren’t enzymes - researchers later
revised the hypothesis: one gene–one protein
• many proteins are composed of several polypeptides
– each of which has its own gene
• can now restated the hypothesis as the one gene–one
polypeptide hypothesis
• **Note: common to refer to gene products as proteins rather
than polypeptides
DNA vs. RNA
Types of RNA
mRNA = messenger RNA
– majority of RNA found in a cell
– carries the genetic information which will be translated into a
protein sequence
– defined by the presence of a “cap” at its 5’ end and a long tail of
adenines at its 3’ end = “poly-A tail”
Types of RNA
rRNA = ribosomal RNA
found in the nucleolus
combines together with the large and small ribosomal subunits to
form the functional ribosome (protein translation)
rRNA is transcribed in the nucleolus by RNA polymerase I
28S rRNA
Types of RNA
tRNA = transfer RNA
actually translates the message coded in the mRNA into a protein
sequence which will become a function protein
tRNA is transcribed in the nucleoplasm by an enzyme called RNA
polymerase III
then exported into the cytoplasm where AA are added
5’
3’
3’
5’
-transcription of RNA is similar to DNA replication – RNA is made in the 5’ to 3’
direction
-enzyme called an RNA polymerase binds to only one of the DNA strands = the
anti-sense (template strand)
-it moves along the template DNA strand (in the 3’ to 5’ direction) and reads the
nucleotide and adds a complementary RNA base
- a growing strand of RNA complementary to the DNA strand results
-BUT rather than a T being paired with an A – U becomes the partner to A
Transcription
-a human gene is also known as a transcription unit = stretch of DNA that is transcribed
into RNA
-a transcription units is comprised of:
1. coding sequence – gives rise to protein strand upon translation
-contains regions of code = “exons” – code for amino acids
-and regions of junk = “introns” – spliced out in the nucleus
5’
3’
Exon
Intron
Exon
Intron
Exon
Intron
Exon
Transcription
-
2. untranslated regions (UTRs) - the regions upstream and downstream of the
coding region that are transcribed but NOT translated into a protein
- -play an important role in translation – can influence the binding of the ribosome
to the mRNA
- -also play a role in exporting the mRNA into the cytoplasm
Transcription
• genes are also associated with additional sequences of DNA
1. core promoter sequence – for the binding of the RNA polymerase
-RNA polymerase recognizes specific sequences of nt’s
-binding is helped out by transcription factors
2. enhancer regions – help enhance transcription
can be several thousands of base pairs upstream of the gene
Transcription
•
•
•
•
the transcription unit is transcribed by an RNA polymerase
three types of RNA polymerase – I, II and III
RNA polymerases create an RNA strand called a primary transcript
• must be modified to produce the final mRNA, tRNA or rRNA
RNA polymerase II transcribes protein coding genes into a primary transcript called premRNA – this is then is processed into mRNA
– genes for tRNA are transcribed in the cytoplasm by RNA polymerase III – primary
transcript is modified into tRNA
– genes for rRNA is transcribed in the nucleolus by RNA polymerase I – primary
transcript is modified into rRNA
-3D representation of the
RNA polymerase II enzyme
Transcription
• three stages of transcription
– Initiation: binding of the RNA polymerase to the
promoter
• special sequences denote this region
– Elongation: movement of the RNA polymerase along
the anti-sense DNA strand and synthesis of the RNA
transcript
– Termination: release of the RNA polymerase from
the DNA
• special sequences denote this region
• differs between prokaryotes and eukaryotes
Promoter
Transcription unit
5
3
Start point
RNA polymerase
DNA
3
5
1. Initiation – RNA polymerase binds to a special sequence
of nucleotides called the promoter
-certain sections of the promoter are important in
1 A eukaryotic promoter
polymerase binding = core promoter
Promoter
Nontemplate strand
DNA
-in prokaryotes the promoter binds the RNA
5
3
5
3
polymerase without help
TATA box
Template strand
Start point
2 Several transcription
-in eukaryotes – the polymerase requires the assistance
Transcription
factors bind to DNA
factors
of proteins called transcription factors
-specific transcription factors bind to the promoter
5
3
3
5
first and then help position the polymerase at the
3 Transcription initiation
promoter
complex forms
-additional transcription factors then bind
RNA polymerase II
Transcription factors
-entire complex is called the Transcription Initiation
5
3
Complex
3
5
3
TAT AAAA
AT AT T T T
5
RNA transcript
Transcription initiation complex
sequence given in texts is that of the sense strand
Promoter
Transcription unit
5
3
Start point
RNA polymerase
DNA
3
5
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
1. Initiation cont…
-RNA polymerase unwinds the DNA helix (acts as a helicase) –
exposes about 10 to 20 nucleotides for copying
-RNA polymerase holds the DNA helix open (acts like the SSBs)
-RNA polymerase initiates RNA synthesis without the need for a
primer
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
2. Elongation – RNA polymerase synthesizes
a complementary RNA strand
-RNA primary transcript grows in the 5’ to 3’
direction
-uses uracil instead of thymine
-the DNA strands reform their helix once the
RNA polymerase moves past the area
-the mRNA strand emerges from the
polymerase-DNA complex
Template strand of DNA
2 Elongation
Rewound
DNA
5
3
3
5
Nontemplate
strand of DNA
RNA nucleotides
3
5
RNA
transcript
RNA
polymerase
A T C C A A
3
C
5
3 end
C A U C C A
5
Multiple RNA polymerases per DNA template
5
T A G G T T
Direction of transcription
Template
strand of DNA
Newly made
RNA
3
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1 Initiation
Nontemplate strand of DNA
3
5
5
3
Unwound
DNA
RNA
transcript
Template strand of DNA
2 Elongation
Rewound
DNA
5
3
3
5
3
5
RNA
transcript
3 Termination
3
5
5
3
5
Completed RNA transcript
3
Direction of transcription (“downstream”)
3. Termination – RNA polymerase
reaches a specific sequence of
nucleotides and stops
transcription
-the RNA polymerase detaches
from the DNA
-the pre-RNA primary transcript is
released
-in prokaryotes – a termination
sequence that detaches the
polymerase
-in eukaryotes – the RNA
polymerase transcribes a
sequence called a polyadenylation signal
– for the release of the pre-RNA
from the polymerase
Transcription
• to modify the primary transcript into mRNA – the
following modifications are made:
– a 5’methylated cap is added to the 5’end
– addition of a 3’ poly A tail
– the coding sequence is “edited” = splicing
Eukaryotic cells modify RNA after transcription
• enzymes in the eukaryotic nucleus modify pre-mRNA before
exporting the mRNA to the cytoplasm
– known as RNA processing
• 5’ methylated cap – plays a role in the docking of the ribosome
to mRNA – for translation
– modified guanine nucleotide added after the transcription of about 20 to 40
nucleotides
5
G
Protein-coding
segment
P P P
5 Cap 5 UTR
Polyadenylation
signal
AAUAAA
Start
codon
Stop
codon
3 UTR
3
AAA … AAA
Poly-A tail
Eukaryotic cells modify RNA after transcription
• 3’ poly A tail – plays a role in the export of the mRNA
into the cytoplasm
– after transcription – an enzyme adds 20 to 250 adenine nucleotides after
the poly-adenylation signal sequence
– also prevents degradation of the mRNA once its in the cytoplasm
5
G
Protein-coding
segment
P P P
5 Cap 5 UTR
Polyadenylation
signal
AAUAAA
Start
codon
Stop
codon
3 UTR
3
AAA … AAA
Poly-A tail
RNA Splicing
•
most eukaryotic genes and pre-RNA transcripts have long noncoding stretches of
nucleotides that lie between coding regions
– the noncoding regions are called intervening sequences, or introns
– coding regions are called exons because they are eventually expressed in the form of a
protein
– RNA splicing removes introns and joins exons, creating an mRNA molecule
with a continuous coding sequence
– the way you splice can also create multiple isoforms from one RNA transcript
5 Exon Intron Exon
Pre-mRNA 5 Cap
Codon
130
31104
numbers
Intron
Exon 3
Poly-A tail
105
146
Introns cut out and
exons spliced together
mRNA 5 Cap
Poly-A tail
1146
5 UTR
Coding
segment
3 UTR
• RNA splicing is carried out by spliceosomes
• Spliceosomes = several proteins and small nuclear
ribonucleoproteins (snRNPs) that recognize specific sequences
found in introns called splice sites
•
snRNPs – found in the nucleus and are made of small nuclear RNA
(snRNA) and proteins
RNA transcript (pre-mRNA)
5
Exon 1
Protein
snRNA
Intron
Exon 2
Other
proteins
snRNPs
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
1. snRNPs and other proteins
combine to form the spliceosome
Spliceosome
5
2. the spliceosome brings the ends
of two exons together
-forms a “lariat” out of the intron
Spliceosome
components
3. the spliceosome cuts the
pre-mRNA and releases the intron
for degradation
5
mRNA
Exon 1
Exon 2
Cut-out
intron
RNA Splicing
•
genes can encode for more than one
protein
–
•
•
DNA
depending on what segments of RNA are
treated as exons and what are treated as
introns during splicing
Exon 1
–
Exon 2
Intron
Exon 3
RNA processing
Translation
Domain 3
cut out a domain – get a different protein
also - exon shuffling may result in the
evolution of new proteins
–
Intron
Transcription
so the way you splice can determine
what proteins eventually get made =
alternative RNA splicing
proteins often are composed of discrete
regions called domains – coded for by
distinct exons
–
•
Gene
introns increase the probability of crossingover between alleles
creates new exon combinations
Domain 2
Domain 1
Polypeptide
Splicing
• for an animation go to
http://sumanasinc.com/webcontent/animatio
ns/content/mRNAsplicing.html
• (don’t worry about the actual proteins!)
Translation
• process of converting an mRNA message into a strand of amino acids that will be
processed into a mature functional protein
• performed by the ribosome in combination with tRNA molecules
• prokaryotes - translation of mRNA can begin before transcription has finished – no
separation between the mRNA and the ribosome
• eukaryotic cell- the nuclear envelope separates transcription from translation
–
mRNA has to be exported out of the nucleus first
DNA
template
strand
5
3
A
C
C
A
A
A
C
C
G
A
G
T
T
G
G
T
T
T
G
G
C
T
C
A
3
5
DNA
molecule
Gene 1
TRANSCRIPTION
Gene 2
U
mRNA
G
G
U
U
U
G
G
C
U
C
5
A
3
Codon
TRANSLATION
Protein
Trp
Phe
Gly
Ser
Gene 3
Amino acid
•
•
•
•
–
•
61 amino acid codons; 3 stop codons
the code is redundant - each amino acid
can be coded for by more than one codon
• e.g. alanine – GCU, GCC, GCA and GCG
• the GC defines the amino acid as alanine
•
in many cases the 3rd codon is important in
defining the amino acid
–
serine –codons are: AGU, AGC
– BUT arginine codons are: AGA and AGG
The Genetic Code
1964
Second mRNA base
A
C
U
UUU
Phe
U
UUC
UAU
UCU
UGU
Tyr
UCC
U
UAC
UGC
C
UCA
UAA Stop
UGA Stop
A
UUG
UCG
UAG Stop
UGG
Trp
G
CUU
CCU
CAU
CGU
Leu
His
A
Cys
Ser
UUA
C
G
CUC
Leu
CAC
CCC
CCA
CUG
CCG
CAG
AUU
ACU
AAU
AUC
Ile
AUG
CAA
Met or
start
GUU
Gln
AAC
ACC
ACA
AAA
ACG
AAG
GCU
GAU
CGA
C
Arg
CGG
Asn
Thr
AUA
CGC
Pro
CUA
U
AGU
G
Ser
AGA
U
C
AGC
Lys
A
Arg
A
AGG
G
GGU
U
Asp
G
GUC
GUA
GUG
GCC
Val
GCA
GCG
GAA
GAG
C
GGC
GAC
Ala
Glu
GGA
GGG
Gly
A
G
Third mRNA base (3 end of codon)
•
How are the instructions for assembling
amino acids into proteins encoded into
DNA?
20 amino acids - only four nucleotide bases
in DNA
how many nucleotides correspond to an
amino acid?
the mRNA nucleotide sequence is “read” in
groups of 3 nucleotides = “codons”
each codon codes for 1 of the 20 amino
acids that make up proteins
called the “genetic code”
First mRNA base (5 end of codon)
•
Molecular Components of Translation
• two components
• 1. transfer RNA (tRNA)
• 2. the ribosome
tRNA
•
•
•
•
tRNA molecule consists of a single RNA strand that is only about 80 nucleotides
long
at one end – anticodon site for the hybridization with the mRNA template
at the other end – attachment site for the amino acid that corresponds to the
mRNA codon
transcribed in the cytoplasm by RNA polymerase III – it folds into its
characteristic shape spontaneously due
to regions that complement each other
3
Amino acid
attachment
site
5
Amino acid
attachment
site
5
3
Hydrogen
bonds
Hydrogen
bonds
A A G
3
Anticodon
(a) Two-dimensional structure
Anticodon
(b) Three-dimensional structure
5
Anticodon
(c) Symbol used
in this book
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P Adenosine
P P P Adenosine
P Pi
ATP
Pi
-amino acids are attached in
the cytoplasm by enzymes
called
aminoacyl-tRNA –synthetases
-one end fits the amino acid,
the other end fits the tRNA
-20 synthetases – each is specific
for only one kind of tRNA
-the tRNA attached to an AA is
called a ‘charged tRNA’
Pi
tRNA
Aminoacyl-tRNA
synthetase
tRNA
Aminoacyl tRNA
(“charged tRNA”)
Amino
acid
P Adenosine
AMP
Computer model
tRNA and the 3rd codon “wobble”
• the tRNA recognizes the codon “triplet” on the mRNA
template
• attached to the tRNA is the amino acid corresponding
to this codon
• there are 61 amino acid codons – so there should be
61 tRNAs
• there are only 45 tRNAs
– some tRNAs can bind more than one codon
• the rules for complementary base pairing at the third
NT of the codon are less stringent
– “flexible” base pairing at this NT = Third Codon Wobble
Ribosomes
• machine of translation
• made in the nucleolus in eukaryotic cells
• comprised of two subunits of proteins (large and small) linked
together with a piece of rRNA
– eukaryotes: 40S small subunit = 33 proteins + 18S rRNA
+ 60S large subunit = 50 proteins + 28S rRNA (+ 5.6S rRNA + 5S rRNA)
– rRNA is transcribed in the nucleolus, proteins are imported from cytoplasm
– everything is assembled in the nucleolus
– subunits are exported out via nuclear pores
– prokaryotic ribosomes and similar but smaller
Ribosomes
• within the large subunit are two sites for the binding of tRNA
– P-site or Peptidyl-tRNA site – “old” AA
– A-site or aminoacyl-tRNA site – incoming AA
• and one E site/Exit site for the exit of the tRNA off the
ribosome
P site (Peptidyl-tRNA
binding site)
Exit tunnel
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
mRNA
binding site
P
A
Large
subunit
Small
subunit
Ribosomes
• eukaryotic ribosomes are similar but are larger vs. prokaryotes
• most evidence now identifies the rRNA as being the catalyst for
the formation of the peptide bond and the growth of the
polypeptide chain
– RNA with enzymatic activity = ribozyme
Growing polypeptide
Amino end
Next amino
acid to be
added to
polypeptide
chain
E
tRNA
mRNA
5
3
Codons
(c) Schematic model with mRNA and tRNA
Building a Polypeptide
• 3 stages of translation:
– Initiation
– Elongation
– Termination
• all three stages require protein “factors”
– called initiation factors or IFs
– in eukaryotes – known as eIFs
1. Initiation of Translation
• the small subunit of the ribosome binds onto the mRNA sequence near the 5’ methylated
cap
• this subunit already has an initiator tRNA (bound to methionine) associated with it
• binding of the small subunit is helped by numerous eukaryotic initiation factors (eIFs)
• the small subunit then glides down the mRNA “scanning” for the first codon - START codon
= AUG (methionine)
-stops so that initiator tRNA can hybridize with the start codon
Large
ribosomal
subunit
3 U A C 5
5 A U G 3
P site
P i
Initiator
tRNA
mRNA
GTP
GDP
E
5
Start codon
mRNA binding site
3
Small
ribosomal
subunit
A
5
Translation initiation complex
3
• once the small subunit is positioned - the large subunit then
assembles and completes the ribosomal “machine”
• helped by even more eIF’s
• the mRNA and the ribsosome form the Translation Initiation
Complex
• the eIF’s are released once this complex forms
• the ribosome is now ready for the next AA - elongation
follows
Large
ribosomal
subunit
3 U A C 5
5 A U G 3
P site
P i
Initiator
tRNA
mRNA
GTP
GDP
E
5
Start codon
mRNA binding site
3
Small
ribosomal
subunit
A
5
Translation initiation complex
3
2. Elongation
of Translation
http://www.youtube.com/watc
h?v=5bLEDd-PSTQ
http://www.youtube.com/watc
h?v=Ikq9AcBcohA
http://www.youtube.com/watc
h?v=NJxobgkPEAo
2. Elongation
of Translation
3. Termination of Translation
Release
factor
Free
polypeptide
5
3
5
2
3
GTP
2
Stop codon
(UAG, UAA, or UGA)
5
GDP 2 P
-translation also stops at specific codons = STOP codons
-UAA, UGA, UAG
-so when the ribosome reaches these sequences – no more AAs are added and the
ribosome detaches from the peptide strand and mRNA
-a release factor cleaves the polypeptide chain from the tRNA and releases it from the
ribosome (GTP hydrolysis)
-the translation machine “breaks apart” – requires an enzyme that uses ATP hydrolysis
3
•
•
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
Completed
polypeptide
Growing
polypeptides
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
(a)
Ribosomes
mRNA
(b)
0.1 m
Mutations
Mutations
Wild type
DNA template strand 3 T A C T T C A A A C C G A T T 5
• small-scale mutations = point
mutations:
• A. nucleotide pair substitutions
– replacement of one NT and its
partner for another pair
• if there is no change to the
eventual codon/amino acid =
silent mutation
• if it changes the amino acid to
a stop codon = nonsense
mutation
• if it changes the amino acid =
missense mutation
5 A T G A A G T
T T G G C T A A 3
mRNA5 A U G A A G U U U G G C U A A 3
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(a) Nucleotide-pair substitution
A instead of G
3 T A C T T C A A A C C A A T T 5
5 A T G A A G T T T G G T T A A 3
U instead of C
5 A U G A A G U U U G G U U A A 3
Met
Lys
Phe
Gly
Stop
Silent (no effect on amino acid sequence)
T instead of C
3 T A C T T C A A A T C G A T T 5
5 A T G A A G T T T A G C T A A 3
A instead of G
5 A U G A A G U U U A G C U A A 3
Met
Lys
Phe
Ser
Stop
Missense
A instead of T
3 T A C A T C A A A C C G A T T 5
5 A T G T A G T T T G G C T A A 3
U instead of A
5 A U G U A G U U U G G C U A A 3
Met
Nonsense
Stop
Mutations
(b) Nucleotide-pair insertion or deletion
• B. insertions
• C. deletions
• insertions and
deletions can lead to
a frame-shift
• alters how the codons
are read downstream
from the mutation
Extra A
5
3
T
A
C
A
T
T
C
A
A
A
C
C
G
A
T
T
5
A
T
G
T
A
A
G
T
T
T
G
G
C
T
A
A 3
A
A
G
U
U
U
G
G
C
U
A
A 3
Extra U
5
A
U
G
U
Met
Stop
Frameshift causing immediate nonsense
(1 nucleotide-pair insertion)
missing
A
3
T
A
5
A
T
C
T
T
G
A
A
C
A
A
C
C
G
G
T
T
G
G
C
A
T
T
A
T 5 T
A 3A
U
A
A
U missing
5
A
U
G
A
A
G
U
Lys
Met
U
G
G
Leu
C
3
Ala
Frameshift causing extensive missense
(1 nucleotide-pair deletion)
T
T
C
missing
3
T
A
C
A
A
5
A
T
G
T
T
A A
5
A
U
Met
G
G
U
A
C
C
G
A
T
T 5
T
G
G
C
T
A
A 3
G
C
U
A
A 3U
missing
U
Phe
U
G
Gly
No frameshift, but one amino acid missing
(3 nucleotide-pair deletion)
Stop
A
A