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PROTEIN

SYNTHESIS

PROTEIN SYNTHESIS

DNA: NUCLEIC
ACID, DOUBLE
STRAND, PO4, DEOXYRIBOSE SUGAR.
BASE PAIRS (N)
T=THYMINE
A=ADENINE
C= CYTOSINE
G=GUANINE

RNA: NUCLEIC
ACID, SINGLE
STRAND, PO4,
RIBOSE SUGAR.
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

transcription into pre-mRNA


poly-A
tail

cap
5

3
(snipped out)

(snipped out)

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

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

ribosomal
subunits

mature
tRNA

Convergence
of RNAs
Cytoplasmic
pools of
amino acids,
tRNAs, and
ribosomal
subunits

Synthesis of a
polypetide chain at
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

GLUTAMATE

HISTIDINE

GLUTAMATE

GLUTAMATE

VALINE

PROLINE
LEUCINE
HISTIDINE

THREONINE

VALINE

mRNA transcribed from the DNA

PART OF PARENTAL DNA TEMPLAT

ARGININE

GLYCINE

TYROSINE

ARGININE

GLYCINE

LEUCINE

TRYPTOPHAN

LEUCINE

ASPARAGINE

resulting amino acid sequence

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.
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

tRNA MOLECULE

amino acid attachment site

amino
acid

amino acid
attachment site
OH

The structure of transfer RNA (tRNA)

B. Transfer RNA (tRNA)


amino acid
attachment site

methionine

U A

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

Proteins
C.30
C.50

5.8 S
70 S

Mitochondrion 78 S
(prokaryotic
ribosome)

30 S

25 S
16 S

C. 24

50 S

4.5 S

C. 35

5 S
30 S

23 S
18 S

C. 33

50 S

5S

C. 35

26 S

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)

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.
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ASK - 2009

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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
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ASK - 2009

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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
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ASK - 2009

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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
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ASK - 2009

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Initiation
aa1

1-tRNA
anticodon
hydrogen
bonds

U A C
A U G
codon

aa2

2-tRNA

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

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ASK - 2009

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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

09/29/16

ASK - 2009

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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
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40
ASK - 2009

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
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ASK - 2009

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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

aa2

aa4

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.
aa5
aa4
aa3
aa2

aa199

aa1

aa200

Polyribosome

Groups of ribosomes reading same mRNA simultaneously


producing many proteins (polypeptides).

incoming
large
subunit
1

incoming
small subunit

polypeptide

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:
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
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

The other DNA


molecule carries
a gene mutation

GLUTAMATE

PROLINE

VALINE
HISTIDINE

LEUCINE

THREONINE

VALINE

A summary of transcription and translation in a eukaryotic cell