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PROTEIN SYNTHESIS

PROTEIN SYNTHESIS
DNA: NUCLEIC RNA: NUCLEIC
ACID, DOUBLE ACID, SINGLE
STRAND, PO4, DE- STRAND, PO4,
OXYRIBOSE SUGAR. RIBOSE SUGAR.

BASE PAIRS (N) BASE PAIRS (N)


T=THYMINE U = URACIL
A=ADENINE A=ADENINE
C= CYTOSINE C=CYTOSINE
G=GUANINE G=GUANINE
URACIL (U)
base with a single-ring structure

phosphate
group

sugar (ribose)
POINTS ABOUT
TRANSCRIPTION
NEED RNA POLYMERASE
CODES FOR 20 AMINO ACIDS
CODON:SERIES OF TRIPLET BASE
PAIRS.
64 CODONS, 60 FOR AA, OTHERS
FOR STARTS/STOPS.
INTRONS=NON-CODING
EXONS= CODING FOR RNA
PROTEIN TRANSCRIPTION
NUCLEUS
RNA POLYMERASE CODES TO DNA
DNA TRANSCRIBES TO m-RNA
INTRONS SNIPPED OUT
EXONS KEPT IN CODE
unit of transcription in a DNA strand
exon intron exon intron exon
3 5

transcription into pre-mRNA

poly-A
cap tail
5 3

(snipped out) (snipped out)

5 3
mature mRNA transcript
transcribed DNA DNA to be
winds up again transcribed
sugar-phosphate backbone of one strand
unwinds
of nucleotides in a DNA double helix

Newly forming The DNA


RNA transcript template at the
assembly site

sugar-phosphate part of the


backbone of sequence of growing RNA transcript
the other strand base pairs in DNA 3
5
of nucleotides

3 5
direction of transcription

5 3

RNA polymerase
PROTEIN TRANSLATION
m-RNA GOES THRU 20 TYPES OF AA
RIBOSOME. ANTICODON ON
RIBOSOME IS r- ONE END OF t-
RNA,CODE THREADS RNA.
THRU RIBOSOME. AA ON OTHER END
AREA OF RIBOSOME OF t-RNA
BOUND TO tRNA AA ATTACH TO
EACH OTHER IN
PEPTIDE BOND
FORM PROTEINS
Binding site for mRNA

P
(first
binding
site for A
tRNA) (second
binding
site for
tRNA)
TRANSCRIPTION Unwinding of gene regions of a DNA molecule

Pre mRNA
Transcript mRNA rRNA tRNA
Processing

protein
subunits
Mature mRNA ribosomal mature
transcripts subunits tRNA

Convergence
TRANSLATION of RNAs

Cytoplasmic
pools of
amino acids,
tRNAs, and
Synthesis of a ribosomal
polypetide chain at subunits
binding sites for
mRNA and tRNA
on the surface of
an intact ribosome

FINAL PROTEIN
Destined for use in
cell or for transport
VALINE PROLINE THREONINE

LEUCINE
HISTIDINE GLUTAMATE GLUTAMATE

VALINE PROLINE THREONINE VALINE

LEUCINE
HISTIDINE GLUTAMATE
mRNA transcribed from the DNA

PART OF PARENTAL DNA TEMPLATE

ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE resulting amino acid sequence

ARGININE GLYCINE LEUCINE LEUCINE GLUTAMATE

altered message in mRNA

A BASE INSERTION (RED) IN


DNA

the altered amino acid


sequence
Overview: the roles of transcription and translation in the flow of
genetic information
The triplet code
TRANSCRIPTION AND
TRANSLATION
C DNA. ATC-GCG-TAT
m-RNA. UAG-CGC-AUA
t-RNA. AUC-GCG-UAU
AMINO ACID ISO-ALA-TYR

PEPTIDE BONDS/POLYPEPTIDES/PROTEINS
Translation

Nuclear
DNA membrane

Transcription
Pre-mRNA

Eukaryotic RNA Processing

Cell mRNA

Ribosome

Translation

Protein
Translation
Synthesis of proteins in the cytoplasm

Involves the following:


1. mRNA (codons)
2. tRNA (anticodons)
3. rRNA
4. ribosomes
5. amino acids
Types of RNA
Three types of RNA:
A. messenger RNA (mRNA)
B. transfer RNA (tRNA)
C. ribosome RNA (rRNA)

Remember: all produced in the nucleus!


A. Messenger RNA (mRNA)
Carries the information for a specific protein.
Made up of 500 to 1000 nucleotides long.
Made up of codons (sequence of three bases:
AUG - methionine).
Each codon, is specific for an amino acid.
A. Messenger RNA (mRNA)
start
codon

mRNA A U G G G C U C C A U C G G C G C A U A A

codon 1 codon 2 codon 3 codon 4 codon 5 codon 6 codon 7

protein methionine glycine serine isoleucine glycine alanine stop


codon

Primary structure of a protein


aa1 aa2 aa3 aa4 aa5 aa6

peptide bonds
B. Transfer RNA (tRNA)
Made up of 75 to 80 nucleotides long.
Picks up the appropriate amino acid floating in
the cytoplasm (amino acid activating enzyme)
Transports amino acids to the mRNA.
Have anticodons that are complementary to
mRNA codons.
Recognizes the appropriate codons on the
mRNA and bonds to them with H-bonds.
anticodon

codon in mRNA
anticodon

amino acid
attachment site
amino
tRNA MOLECULE acid OH

amino acid attachment site


The structure of transfer RNA (tRNA)
B. Transfer RNA (tRNA)
amino acid
attachment site methionine amino acid

U A C
anticodon
C. Ribosomal RNA
(rRNA)
Made up of rRNA is 100 to 3000 nucleotides long.

Important structural component of a ribosome.


Associates with proteins to form ribosomes.
Ribosomes
Large and small subunits.
Composed of rRNA (40%) and proteins
(60%).
Both units come together and help bind the
mRNA and tRNA.
Two sites for tRNA
a. P site (first and last tRNA will attach)
b. A site
Ribosomes
Origin Complete Ribosomal rRNA Proteins
ribosome subunit components
Cytosol 80 S 40 S 18 S C.30
(eukaryotic 60 S 5S C.50
ribosome) 5.8 S
25 S
Chloroplasts 70 S 30 S 16 S C. 24
(prokaryotic 50 S 4.5 S C. 35
ribosome) 5 S
23 S
Mitochondrion 78 S 30 S 18 S C. 33
(prokaryotic 50 S 5S C. 35
ribosome) 26 S
Ribosomes

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G

Small subunit
Translation
Three parts:
1. initiation: start codon (AUG)
2. elongation:
3. termination: stop codon (UAG)

Lets make a PROTEIN!!!!.


Translation

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G

Small subunit
Translation
Initiation
The inactive 40S and 60S subunits will bind to
each other with high affinity to form inactive
complex unless kept apart
This is achieved by eIF3, which bind to the
40S subunit
mRNA forms an initiation complex with a
ribosome
A number of initiation factors participate in
the process.
33
Translation
Cap sequence present at the 5 end of the
mRNA is recognized by eIF4
Subsequently eIF3 is bound and cause the
binding of small 40S subunit in the complexes
The 18S RNA present in the 40 S subunit is
involved in binding the cap sequence
eIF2 binds GTP and initiation tRNA, which
recognize the the start codon AUG
This complex is also bound to 40S subunit
34
Translation
Driven by hydrolysis of ATP, 40S complex
migrate down stream until it finds AUG start
codon
The large 60S subunit is then bound to the 40S
subunit
It is accompanied by the dissociation of several
initiation factor and GDP
The formation of the initiation complex is now
completed
Ribosome complex is able to translate

35
Translation
Extrachromosomal mRNAs have no cap site
Plastid mRNA has a special ribosome binding site for
the initial binding to the small subunit of the ribosome
(shine-Dalgarno sequence)
This sequence is also found in bacterial mRNA, but it is
not known in the mitochondria
In the prokaryotic, the initiation tRNA is loaded with
N-formylmethionine
After peptide formation, the formyl residue is cleaved
from the methionine

36
Initiation
aa2
aa1

2-tRNA
1-tRNA
G A U
anticodon U A C
hydrogen A U G C U A C U U C G A
bonds codon mRNA
Translation
Elongation
A ribosome contains two sites where the
tRNAs can bind to the mRNA.
P (peptidyl) site allows the binding of the
initiation tRNA to the AUG start codon.
The A (aminoacyl) site covers the second
codon of the gene and the first is
unoccupied
On the other side of the P site is the exit
(E) site where empty tRNA is released

38
Translation
Elongation
The elongation begins after the corresponding
aminoacyl-tRNA occupies the A site by forming
base pairs with the second codon
Two elongation factors (eEF) play an important
role
eEF1 binds GTP and guides the corresponding
aminoacyl-tRNA to the A site, during which GTP is
hydrolized to GDP and P.
The cleavage of the energy-rich anhydride bond in
GTP enables the aminoacyl-tRNA to bind to codon
at the A site

39
Translation
Elongation
Afterwards the GDP still bound to eEF1, is exchange
for GTP as mediated by the eEF1
The eEF1 -GTP is now ready for the next cycle
Subsequently a peptide linkage is form between the
carboxyl group of methionine and the amino group of
amino acid of the tRNA bound to A site
Peptidyl transferase catalyzing the reaction. It
facilitates the N-nucleophilic attack on the carboxyl
group, whereby the peptide bond is formed with the
released of water
40
Translation
Elongation
Accompanied by the hydrolysis of one molecule
GTP to form GDP and P, the eEF2 facilitates
the translocation of the ribosome along the
mRNA to three bases downstream
Free tRNA arrives at site E is released, and
tRNA loaded with the peptide now occupies the
P Site
The third aminoacyl-tRNA binds to the vacant
A site and a further elongation cycle can begin
41
Elongation
peptide bond
aa3
aa1 aa2

3-tRNA

1-tRNA 2-tRNA G A A
anticodon U A C G A U
hydrogen A U G C U A C U U C G A
bonds codon mRNA
aa1 peptide bond
aa3
aa2

1-tRNA

U A C 3-tRNA
(leaves)
2-tRNA G A A

G A U
A U G C U A C U U C G A
mRNA

Ribosomes move over one codon


peptide bonds
aa1 aa4

aa2 aa3

4-tRNA

2-tRNA 3-tRNA G C U

G A U G A A
A U G C U A C U U C G A A C U
mRNA
peptide bonds
aa1 aa4
aa2

aa3

2-tRNA
4-tRNA
G A U
(leaves) 3-tRNA G C U

G A A
A U G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon


peptide bonds aa5
aa1
aa2
aa4
aa3

5-tRNA

U G A
3-tRNA 4-tRNA

G A A G C U
G C U A C U U C G A A C U
mRNA
aa1 peptide bonds aa5
aa2
aa3
aa4

5-tRNA

3-tRNA U G A
G A A 4-tRNA

G C U
G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon


aa5
aa4 aa199 Termination
aa3 primary aa200
structure
aa2 of a protein

aa1
terminator
200-tRNA
or stop
codon
A C U C A U G U U U A G
mRNA
Translation
Release
When A site finally binds to a stop codon (UGA,
UAG, UAA)
Stop codons bind eRF accompanied by
hydrolysis GTP to form GDP and P
Binding of eRF to the stop codon alters the
specificity the peptidyl transferase
Water instead amino acid is now the acceptor
for the peptide chain
Protein released from the tRNA
Translation
The difference
Eukaryotic and prokaryotic translation can react
differently to certain antibiotics
Puromycin
an analog tRNA and a general inhibitor of protein
synthesis
Cycloheximide
only inhibits protein synthesis by eukaryotic ribosomes
Chloramphenicol, Tetracycline, Streptomycin
inhibit protein synthesis by prokaryotic ribosome
End Product
The end products of protein synthesis is a
primary structure of a protein.

A sequence of amino acid bonded together by


peptide bonds.
aa5
aa3 aa4
aa2 aa199

aa1 aa200
Polyribosome
Groups of ribosomes reading same mRNA simultaneously
producing many proteins (polypeptides).

incoming
large
subunit

1 2 3 4 5 6 7
mRNA

incoming
small subunit polypeptide
TYPES OF PROTEINS
ENZYMES/HELICASE
CARRIER/HEMOGLOBIN
IMMUNOGLOBULIN/ANTIBODIES
HORMONES/STEROIDS
STRUCTURAL/MUSCLE
IONIC/K+,Na+
all regulate things put together critter
Protein Sorting
Vast majority of protein within the cell are synthesized
within the cytoplasm, but the final sub-cellular location
can be in one of a whole array of membrane-bound
compartment
Protein is subjected to be sorted for special targeted
organelles
Protein Sorting
Vast majority of protein within the cell are synthesized
within the cytoplasm, but the final sub-cellular location
can be in one of a whole array of membrane-bound
compartment
Protein is subjected to be sorted for special targeted
organelles:
Plastids
Mitochondria
Peroxisomes
Vacuoles
Mitochondria
More than 95% of mitochondrial proteins in plant are encoded in the
nucleus and translated in the cytosol
Proteins are generally equipped with targeting signals ( a signal
sequence of 12-70 amino acids at the amino terminal)
Protein import occurs at translocation site
In most cases, protein destined for the mitochondrial inner
membrane after transport through outer membrane are guided
directly to the location by internal targeting sequence
Protein destined for the inner mitochondrial membrane contain pro-
sequence that guides first into the mitochondrial matrix. After
removal of the pro-sequence by processing peptidase, the proteins
are directed by second targeting signal sequence into the inner
membrane
Plastids
ATP is consumed for the phosphorilation of a protein,
probably the receptor OEP86
The protein transport is regulated by the binding of the
GTP to OEP86 and OEP34
After the protein is delivered, the pre-sequence is
removed by a processing peptidase
The protein destined to thylakoid membrane are first
delivered into stroma and then directed by internal
targeting signal into thylakoid membrane
Peroxisomes
Small membrane-bound cytoplasmic organelle
containing oxidizing enzymes
They can be found in leaf cells where they contain
some of the enzymes of glycolytic pathway
All protein have to be delivered from the cytosol
The transport is accompanied by ATP hydrolysis
Targeting sequence SKL (serine-lysine-leucine) has
been observed in C terminus, but this sequence is not
removed after uptake
Vacuole
Proteins are transferred during their synthesis to the lumen of ER
This is aided by a signal sequence at the terminus of the
synthesized protein, which binds with a signal recognition particle
to a pore protein present in the ER membrane and thus directs the
protein to the ER lumen
In such cases, ribosome is attached to the ER membrane during
protein synthesis and the synthesized protein appears immediately
in the ER lumen. It is called co-translational protein transport
This protein is then transferred from the ER by vesicles transfer
across the golgi apparatus to the vacuole or are exported by
secretory vesicles from the cell
Coupled transcription and translation in bacteria
original a base
base triplet substitution
in a DNA within the
strand triplet (red)
As DNA is replicated, proofreading
enzymes detect the mistake and
make a substitution for it:

POSSIBLE OUTCOMES:

OR

One DNA molecule The other DNA


carries the original, molecule carries
unmutated sequence a gene mutation

VALINE PROLINE THREONINE VALINE

LEUCINE
HISTIDINE GLUTAMATE
A summary of transcription and translation in a eukaryotic cell