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PROTEIN SYNTHESIS
PROTEIN SYNTHESIS
 DNA: NUCLEIC
ACID, DOUBLE
STRAND, PO4, DEOXYRIBOSE SUGAR.
 RNA: NUCLEIC
ACID, SINGLE
STRAND, PO4,
RIBOSE SUGAR.
 BASE PAIRS (N)
 T=THYMINE
 A=ADENINE
 C= CYTOSINE
 G=GUANINE
 BASE PAIRS (N)
 U = URACIL
 A=ADENINE
 C=CYTOSINE
 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
exon
unit of transcription in a DNA strand
intron
exon
intron
exon
3’
5’
transcription into pre-mRNA
poly-A
tail
cap
5’
3’
(snipped out)
(snipped out)
5’
3’
mature mRNA transcript
sugar-phosphate backbone of one strand
of nucleotides in a DNA double helix
sugar-phosphate
backbone of
the other strand
of nucleotides
part of the
sequence of
base pairs in DNA
transcribed DNA
winds up again
DNA to be
transcribed
unwinds
Newly forming
RNA transcript
The DNA
template at the
assembly site
growing RNA transcript
5’
3’
5’
RNA polymerase
direction of transcription
3’
5’
3’
PROTEIN TRANSLATION
 m-RNA GOES THRU
RIBOSOME.
 RIBOSOME IS rRNA,CODE THREADS
THRU RIBOSOME.
 AREA OF RIBOSOME
BOUND TO tRNA
 20 TYPES OF AA
 ANTICODON ON
ONE END OF tRNA.
 AA ON OTHER END
OF t-RNA
 AA ATTACH TO
EACH OTHER IN
PEPTIDE BOND
 FORM PROTEINS
Binding site for mRNA
P
(first
binding
site for
tRNA)
A
(second
binding
site for
tRNA)
TRANSCRIPTION
Pre mRNA
Transcript
Processing
Unwinding of gene regions of a DNA molecule
mRNA
rRNA
tRNA
protein
subunits
Mature mRNA
transcripts
TRANSLATION
Synthesis of a
polypetide chain at
binding sites for
mRNA and tRNA
on the surface of
an intact ribosome
ribosomal
subunits
mature
tRNA
Convergence
of RNAs
Cytoplasmic
pools of
amino acids,
tRNAs, and
ribosomal
subunits
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
ARGININE
GLYCINE
LEUCINE
LEUCINE
GLUTAMATE
resulting amino acid sequence
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.
 m-RNA.
 t-RNA.
 AMINO ACID
ATC-GCG-TAT
UAG-CGC-AUA
AUC-GCG-UAU
ISO-ALA-TYR
 PEPTIDE BONDS/POLYPEPTIDES/PROTEINS
Translation
Nuclear
membrane
DNA
Transcription
Eukaryotic
Cell
Pre-mRNA
RNA Processing
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
protein methionine
codon 2
codon 3
glycine
serine
codon 4
isoleucine
codon 5
codon 6
glycine
alanine
codon 7
stop
codon
Primary structure of a protein
aa1
aa2
aa3
peptide bonds
aa4
aa5
aa6
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
tRNA MOLECULE
amino acid attachment site
amino
acid
OH
The structure of transfer RNA (tRNA)
B. Transfer RNA (tRNA)
amino acid
attachment site
methionine
U A
C
anticodon
amino acid
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
Cytosol
(eukaryotic
ribosome)
Chloroplasts
(prokaryotic
ribosome)
Complete Ribosomal
ribosome subunit
80 S
40 S
60 S
rRNA
components
18 S
5S
5.8 S
25 S
Proteins
70 S
30 S
50 S
16 S
4.5 S
5 S
23 S
C. 24
C. 35
 30 S
 50 S
18 S
5S
26 S
C. 33
C. 35
Mitochondrion 78 S
(prokaryotic
ribosome)
C.30
C.50
Ribosomes
Large
subunit
P
Site
A
Site
mRNA
A U G
Small subunit
C U A C U U C G
Translation
 Three parts:
1. initiation: start codon (AUG)
2. elongation:
3. termination: stop codon (UAG)
 Let’s make a PROTEIN!!!!.
Translation
Large
subunit
P
Site
A
Site
mRNA
A U G
Small subunit
C U A C U U C G
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
aa1
aa2
2-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
G A U
C U A C U U C G A
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
anticodon
hydrogen
bonds
U A C
A U G
codon
2-tRNA
G A A
G A U
C U A C U U C G A
mRNA
aa1
peptide bond
aa3
aa2
1-tRNA
3-tRNA
U A C
(leaves)
2-tRNA
A U G
G A A
G A U
C U A C U U C G A
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa4
aa2
aa3
4-tRNA
2-tRNA
A U G
3-tRNA
G C U
G A U G A A
C U A C U U C G A A C U
mRNA
aa1
peptide bonds
aa4
aa2
aa3
2-tRNA
4-tRNA
G A U
(leaves)
3-tRNA
A U G
G C U
G A A
C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa5
aa2
aa3
aa4
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
peptide bonds
aa1
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
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
aa4
aa5
Termination
aa199
aa3 primary
structure
aa2 of a protein
aa200
aa1
200-tRNA
A C U
mRNA
terminator
or stop
codon
C A U G U U U A G
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.
aa2
aa1
aa3
aa4
aa5
aa199
aa200
Polyribosome
• Groups of ribosomes reading same mRNA simultaneously
producing many proteins (polypeptides).
incoming
large
subunit
1
incoming
small subunit
2
3
4
polypeptide
5
6
7
mRNA
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 prosequence 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
base triplet
in a DNA
strand
a base
substitution
within the
triplet (red)
As DNA is replicated, proofreading
enzymes detect the mistake and
make a substitution for it:
POSSIBLE OUTCOMES:
OR
One DNA molecule
carries the original,
unmutated sequence
VALINE
PROLINE
The other DNA
molecule carries
a gene mutation
THREONINE
VALINE
LEUCINE
HISTIDINE
GLUTAMATE
A summary of transcription and translation in a eukaryotic cell