Download Lecture 8: Life`s Information Molecule III

BIO 2, Lecture 8
LIFE’S INFORMATION
MOLECULE III: TRANSLATION AND
PROTEIN LOCALIZATION
• In addition to mRNAs, genes can also
code for other types of RNAs
• These other RNAs do not code for
proteins
– Examples: transfer RNA (tRNA),
ribosomal RNA (rRNA)
• These RNAs fold into secondary and
tertiary structures and perform
important functions in the cell (much
like proteins)
mRNA
transcription
translation
mRNA
translation
tRNA
transcription
rRNA
One or more
polypeptides
One or more
polypeptides
help with
tRNAs
• tRNAs are short RNA molecules (~80
bases) that fold back upon themselves
to form a cloverleaf-shaped structure
• Molecules of tRNA are not identical:
– Each carries a specific amino acid (and
will only carry that amino acid)
– Each has an anticodon on the other end
that recognizes and base-pairs with a
complementary codon on mRNA
3
Amino acid
attachment site
5
Hydrogen
bonds
Anticodon
(a) Two-dimensional structure
Amino acid
attachment site
5
3
Hydrogen
bonds
3
Anticodon
(b) Three-dimensional structure
Anticodon
5
(c) Symbol used
in this book
Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
tRNA
Anticodon
Codons
5
mRNA
3
• Accurate translation requires two steps:
– 1. An enzyme called aminoacyl-tRNA
synthetase adds an amino acid to all the
tRNAs that carry the anticodon that is
complementary to the codon in the mRNA
that codes for that amino acid
– Second: The tRNA anticodon recognizes and
base-pairs to its 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
P P P
ATP
Adenosine
Aminoacyl-tRNA
synthetase (enzyme)
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P
Adenosine
ATP
P
P Pi
Pi
P
i
Adenosine
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P
Adenosine
ATP
P
P Pi
Pi
P
i
Adenosine
tRNA
Aminoacyl-tRNA
synthetase
tRNA
P
Adenosine
AMP
Computer model
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P
Adenosine
ATP
P
P Pi
Pi
P
Adenosine
tRNA
Aminoacyl-tRNA
synthetase
i
tRNA
P
Adenosine
AMP
Computer model
Aminoacyl-tRNA
(“charged tRNA”)
• Ribosomes facilitate specific coupling of
tRNA anticodons with mRNA codons in
protein synthesis
• The two ribosomal subunits (large and
small) are made up of a combination of
proteins and ribosomal RNA (rRNA)
30 S (small) subunit of the bacterial ribosome
Proteins = blue, RNA = pink
tRNA
molecules
Growing
polypeptide
Exit tunnel
Large
subunit
EPA
Small
subunit
5’
mRNA
3’
(a) Computer model of functioning ribosome
• 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 tRNAs
(which have now lost their amino acids) leave
the ribosome
P site (Peptidyl-tRNA
binding site)
E site
(Exit site)
A site (AminoacyltRNA binding site)
E P A
mRNA
binding site
Large
subunit
Small
subunit
(b) Schematic model showing binding sites
Growing polypeptide
Amino end
Next amino acid
to be added to
polypeptide chain
mRNA
5
E
tRNA
3
Codons
(c) Schematic model with mRNA and tRNA
• The three stages of translation:
– Initiation
– Elongation
– Termination
• Requires energy (in the form of GTP) and
additional proteins (called “factors”)
• In prokaryotes, initiation takes place
when an rRNA in the small ribosomal
subunit base-pairs to a region in the
5’UTR of the mRNA
• Then the small subunit moves along the
mRNA (through the 5’ UTR) until it
reaches the start codon (always 5’AUG-3’)
• Proteins called initiation factors bring
in the large subunit that completes the
translation initiation complex
3’‘U A C 5’
5’A U G 3’
Initiator
tRNA
mRNA
5’
P site
GTP
Start codon
mRNA binding site
Large
ribosomal
subunit
3’
Small
ribosomal
subunit
GDP
E
5’
A
3’
Translation initiation complex
• 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 end
of polypeptide
mRNA
5
E
3
P
A
site site
Amino end
of polypeptide
mRNA
5
E
3
P
A
site site
GTP
GDP
E
P A
Amino end
of polypeptide
mRNA
5
E
3
P
A
site site
GTP
GDP
E
P A
E
P A
Amino end
of polypeptide
mRNA
Ribosome ready for
next aminoacyl tRNA
E
3
P
A
site site
5
GTP
GDP
E
E
P A
P A
GDP
GTP
E
P A
• 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
Release
factor
3’
5’
Stop codon
(UAG, UAA, or UGA)
Release
factor
Free
polypeptide
3’
5’
5’
Stop codon
(UAG, UAA, or UGA)
3’
2 GTP
2 GDP
Release
factor
Free
polypeptide
5’
3’
5’
5’
Stop codon
(UAG, UAA, or UGA)
3’
2 GTP
2 GDP
3’
• In both prokaryotes and eukaryotes, 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 from a single mRNA
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5’ end)
(a)
End of
mRNA
(3’ end)
Ribosomes
mRNA
(b)
0.1 µm
• Remember, though, that translation is
not sufficient to make a protein (which is
a functional molecule)
• Polypeptide chains are modified after
translation and then targeted to the
correct location in the cell before they
can fold and, if the protein has
quarternary structure, get together with
other polypeptides, to function properly
• Two populations of ribosomes are evident in
eukaryotic 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
ENDOPLASMIC RETICULUM (ER)
Flagellum
Rough ER
Smooth ER
Nuclear
envelope
Nucleolus
NUCLEUS
Chromatin
Centrosome
Plasma
membrane
CYTOSKELETON:
Microfilaments
Intermediate
filaments
Microtubules
Ribosomes
Microvilli
Golgi
apparatus
Peroxisome
Mitochondrion
Lysosome
Nucleus
Rough ER
Smooth ER
cis Golgi
trans Golgi
Plasma
membrane
• 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
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
SRP
receptor
protein
Translocation
complex
ER
membrane
Protein
• Mutations are changes in the sequence,
number of copies, or location of a gene
• The smallest type of mutation is a point
mutation
• Involves a very small change within a single
gene
• Can involve the substitution of one base-pair
for another OR the deletion or insertion of a
small number of nucleotide pairs
• Even though they are small, can cause major
changes to the function of a protein
• Point mutations come in two main types
including:
• Single Base-pair substitutions
• Silent
• Missense
• Nonsense
• Insertions or deletions
• Frameshift leading to immediate nonsense
• Frameshift leading to extensive missense
• Missing amino acid(s)
• 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
Wild type
DNA template 3’
strand 5’
5’
3’
mRNA 5’
3’
Protein
Stop
Amino end
Carboxyl end
A instead of G
5’
3’
3’
5’
U instead of C
5’
3’
Stop
Silent (no effect on amino acid sequence)
Wild type
DNA template 3’
strand 5’
5’
3’
mRNA 5’
3’
Protein
Stop
Amino end
Carboxyl end
T instead of C
5
3
3
5
A instead of G
3
5
Stop
Missense
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
A instead of T
3
5
5
3
U instead of A
5
3
Stop
Nonsense
• Insertions and deletions are additions
or losses of nucleotide pairs in a gene
• These mutations have a disastrous
effect on the resulting protein even
more often than substitutions do
• Insertion or deletion of nucleotides
may alter the reading frame, producing
a frameshift mutation – most of which
eliminate the protein’s functional
altogether
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
Extra A
5
3
3
5
Extra U
5
3
Stop
Frameshift causing immediate nonsense (1 base-pair insertion)
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
missing
5
3
3
5
missing
5
3
Frameshift causing extensive missense (1 base-pair deletion)
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
missing
5
3
3
5
missing
5
3
Stop
No frameshift, but one amino acid missing (3 base-pair deletion)