Protein Synthesis

Dian Purwita Sari, M.Biotech., Apt.

FKIK 2012

Reference

All life depends on three critical

molecules

DNAs
Hold information on how cell works
RNAs
Act to transfer short pieces of information to
different parts of cell
Provide templates to synthesize into protein
Proteins
Form enzymes that send signals to other cells
and regulate gene activity
Form bodys major components (e.g. hair, skin,
etc.)
3

DNA

RNA

PROTEIN

Genome : 40.000 genes

Transcriptome >
100.000 RNA
Variability :
genomic variations

Proteome > 400.000

proteins

Gene expression,
alternative splicing
Protein
cleavage,modification
4

DNA

DNA

Located in 23 pairs of
chromosomes in
nucleus of cell
DNA has two
functions:

Replication reproduces itself

when cell divides
Information
transmission
via protein
synthesis
From: Tortora, GJ & Grabowski SR (2000) Principles of Anatomy
and Physiology (9th Ed). New York: John Wiley & Sons.
P86.

DNA

DNA contains
genetic information

Gene - segment of DNA

on a chromosome that
codes for a particular
protein
Coding contained in
sequence of bases (on
mRNA) which code for a
particular amino acid
(i.e. genetic code)
Genetic code universal
in all organisms
Mitochondrial DNA
slightly different

From: Elliott WH & Elliott DC. (1997) Biochemistry and Molecular Biology. New
York: Oxford University Press. P294.

Gene Expression
Gene expression is the
process by which a genes
information is converted into
the structures and functions
of a cell by a process of
producing a biologically
functional molecule of either
protein or RNA (gene product)
is made.
Gene expression is
assumed to be controlled at
various points in the sequence
leading to protein synthesis.

Information transmission

Information stored in DNA transferred

to RNA and then expressed in the
structure of proteins

Two steps in process:

Transcription - information transcribed from DNA

into mRNA
Translation - information in mRNA translated into
primary sequence of a protein

Protein Synthesis
Protein Synthesis

synthesis of an RNA
(mRNA) that is
complementary to one of
the strands of DNA.

is the process in which

cells build proteins from
information in a DNA
gene in a two major
steps:

ITranscription and
II-Translation

Transcription :

Translation :

ribosomes read a
messenger RNA and
make protein according
to its instruction.

The Difference of Procaryote &

Eucaryote

10

Eucaryote Gene Expression Control

nucleus

DNA

Primary
RNA
transcript

transcriptional
control

cytosol

inactive
mRNA
mRNA
degradation
control

mRNA
RNA
processing
control

RNA
transport
control

mRNA
translation
control

protein
nucleus
membrane

protein
activity
control

inactive
11
protein

Transcription

Information transcribed from DNA

into RNA

mRNA carries information for

protein structure, but other RNA
molecules formed in same way

RNA polymerase binds to

promoter nucleotide sequence at
point near gene to be expressed
DNA helix unwinds
RNA nucleotides assemble along
one DNA strand (sense strand) in
complementary sequence to order
of bases on DNA beginning at start
codon (AUG - methionine)
Transcription of DNA sense strand
ends at terminator nucleotide
sequence
mRNA moves to ribosome
DNA helix rewinds
From: Tortora, GJ & Grabowski SR (2000) Principles of Anatomy and
12
Physiology (9th Ed). New York: John Wiley & Sons. P88.

Initiation of transcription:
Promoter = nucleotide sequence in DNA to which RNA
polymerase binds to begin transcription
Cellular signaling

Cytoplasm
Nucleus

RNA polymerase

Pre-mRNA

GENE
Promoter

mRNA processing

13

14

Transcription

16
http://biology.unm.edu/ccouncil/Biology_124/Images/transcription.gif

Transcription

RNA polymerase copies both the exons and the

introns. The stretch of DNA that is transcribed into
an RNA molecule is called a transcription unit.
A transcription unit that is translated into protein
contains coding sequence that is translated into
protein and sequences that direct and regulate
protein synthesis;
Transcription proceeds in the 5' 3' direction.
Transcription is divided into 3 phases: Initiation,
Elongation and Termination..

17

RNA polymerase;

eukaryotic nuclei contain

three RNA polymerases .
RNA polymerase I is
found in the nucleolus;
the other two
polymerases are located
in the nucleoplasm.
The three nuclear RNA
polymerase have
different roles in
transcription.

Polymerase I makes a
large precursor to the
major rRNA (5.8S,18S
and 28S rRNA in
vertebrates).
Polymerase II
synthesizes mRNAs. It
also make most small
nuclear RNAs (snRNAs).
Polymerase III makes
the precursor to
5SrRNA, the tRNAs and
several other small
cellular and viral RNAs.
18

Initiation:
The general transcription factors combine with
RNA polymerase to form a preinitiation
complex that is competent to initiate
transcription as soon as nucleotide are
available.

19

Initiation:

The enzyme RNA polymerase recognizes a

promoter, which lies upstream of the gene. The
polymerase binding causes the unwinding of the
DNA double helix. This is followed by initiation of RNA
synthesis at the starting point.

The RNA polymerase starts building the RNA

chain, it assembles ribonucleotides triphosphates:
ATP; GTP; CTP and UTP into a strand of RNA.

After the first nucleotide is in place, the polymerase

joins a second nucleotide to the first, forming the
initial phosphodiester bond in the RNA chain.
20

RNA
polymerase
directs the sequential
Elongation

binding of riboncleotides to the growing RNA

chain in the 5`-3` direction.

Each ribonucleotide is inserted into the

growing RNA strand following the rules of base
pairing. This process is repeated till the
desired RNA length is
synthesized.

21

Termination
Other regions at the end of genes; called terminators,

Other regions at the end of genes; called terminators,

signal termination. These work in conjunction with RNA
polymerase to loosen the association between RNA
product and DNA template. The result is that the RNA
dissociate from RNA polymerase and DNA and so stop
transcription.

The product is immature RNA or pre mRNA (Primary

transcript).

There are DNA sequence motifs for RNA Pol II

recognition to be terminated.
22

RNA Processing
Pre-mRNA mRNA
Capping: Synthesis of the cap. The 5` cap is a 7methylguanosine (m7G) . The cap protects the mRNA
from being degraded by enzymes; enhancement of
mRNA translatability.

Splicing: Step-by-step removal of introns present in the

pre-mRNA and joining of the remaining exons. The
removal of introns and joining of exons takes place on a
special structures called spliceosomes.

Polyadenylation: Synthesis of the poly (A) tail involves

cleavage of its 3' end and then the addition of about 200
adenine residues to form a poly (A) tail; This completes
the mRNA molecule (mature mRNA), which is now ready
for export to the cytosol for protein synthesis.
23

Splicing

In humans, exons average ~ 200 bp,

introns ~ 10Kbp
Splice sites are indicated by sequence
markers
Alternative splicing
One gene -> Many proteins
Depends on transcription context
tissue, metabolism, etc...
Mechanism of alternative splicing is
not completely understood.
24

Alternative Splicing

Alternative splicing:
is a very common phenomenon in higher
eukaryotes. It is a way to get more than one
protein product out of the same gene and a way to
control gene expression in cells.

Protein isoform
25

Genes to Proteins

26

Translation
Translation is the process by which ribosomes read
the genetic message in the mRNA and produce a
protein product according to the message's
instruction.

27

Translation:FrommRNAtoProtein

process of converting the information in a

sequence of nitrogenous bases in mRNA into a
sequence of amino acids in protein is known as
translation.

takes place at the ribosomes in the cytoplasm.

Involves 3 types of RNA
1. Messenger RNA (mRNA) =carries
the blueprint for construction of a protein
2. Ribosomal RNA (rRNA) =
the construction site where the protein is
made
3. Transfer RNA (tRNA) =
the truck delivering the proper amino acid to
the site at the right time
28

Transfer RNA molecules serve as

interpreters during translation

In the cytoplasm, a
ribosome attaches to
the mRNA and
translates its message
into a polypeptide
The process is aided by
transfer RNAs
Each tRNA molecule has
a triplet anticodon on
one end and an amino
acid attachment site on
the other
Anticodon base pairs
with codon of mRNA
29

tRNA pick up their specific

amino acids from the cytoplasm

30

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32

TRANSLATION
(PROTEIN SYNTHESIS)

Information in mRNA translated into

primary sequence of a protein in 4
steps:

ACTIVATION

INITIATION

ELONGATION

TERMINATION
33

Translation (protein
synthesis)

ACTIVATION

Each amino acid

activated by
reacting with ATP
tRNA synthetase
enzyme attaches
activated amino
acid to own
particular tRNA

Adapted from: Bettelheim FA and March J (1990) Introduction to Organic and

Biochemistry (International Edition). Philadelphia: Saunders College
34
Publishing p398

Translation (protein
synthesis)

INITIATION

mRNA attaches to
smaller body of
ribosome
Initiator tRNA attaches
to start codon
Larger body of
ribosome combines
with smaller body

35 and
From: Tortora, GJ & Grabowski SR (2000) Principles of Anatomy
Physiology (9th Ed). New York: John Wiley & Sons. P88.

Translation (protein
synthesis)

ELONGATION

Anticodon of next tRNA binds

to mRNA codon at A site of
ribosome

Each tRNA specific for one

amino acid only, but some
amino acids coded for by up to
6 codons
Order of bases in mRNA
codons determine which tRNA
anticodons will align and
therefore determines order of
amino acids in protein

Amino acid at A site linked to

previous amino acid
Ribosome moves along one
codon and next tRNA binds at
A site

From: Tortora, GJ & Grabowski SR (2000) Principles of Anatomy and

Physiology (9th Ed). New York: John Wiley & Sons. P88.

36

Translation (protein
synthesis)

TERMINATION

Final codon on mRNA

contains termination
signal
Releasing factors
cleave polypeptide
chain from tRNA that
carried final amino
acid
mRNA released from
ribosome and broken
down into nucleotides
From: Tortora, GJ & Grabowski SR (2000) Principles of Anatomy
37 and
Physiology (9th Ed). New York: John Wiley & Sons. P88.

This is a molecule of messenger RNA.

It was made in the nucleus by
transcription from a DNA molecule.

codon
A U G G G C U U AAAG C AG U G C AC G U U

mRNA molecule
38

A ribosome on the rough endoplasmic

reticulum attaches to the mRNA
molecule.
ribosome
A U G G G C U U AAAG C AG U G C AC G U U

39

Amino acid
tRNA molecule
A transfer RNA molecule arrives.
It brings an amino acid to the first three
bases (codon) on the mRNA.
anticodon

The three unpaired bases (anticodon)

on the tRNA link up with the codon.

U AC
A U G G G C U U AAAG C AG U G C AC G U U

40

Another tRNA molecule comes into

place, bringing a second amino acid.
Its anticodon links up with the second
codon on the mRNA.
CC

G
U AC
A U G G G C U U AAAG C AG U G C AC G U U

41

Peptide bond

A peptide bond forms between the

two amino acids.
U AC C C G
A U G G G C U U AAAG C AG U G C AC G U U

42

The first tRNA molecule releases its amino

acid and moves off into the cytoplasm.

C
A
U

CCG
A U G G G C U U AAAG C AG U G C AC G U U

43

The ribosome moves along the mRNA to

the next codon.
CCG
A U G G G C U U AAAG C AG U G C AC G U U

44

Another tRNA molecule brings

the next amino acid into place.
AA

U
CCG
A U G G G C U U AAAG C AG U G C AC G U U

45

A peptide bond joins the second

and third amino acids to form a
polypeptide chain.

CCGCCG
A U G G G C U U AAAG C AG U G C AC G U U

46

The process continues.

The polypeptide chain gets longer.
This continues until a termination
(stop) codon is reached.
The polypeptide is then complete.

AC

G
GUC
A U G G G C U U AAAG C AG U G C AC G U U

47

Control of protein synthesis

Rate of protein synthesis:

suppressed during exercise

increases for up to 48 hours post-exercise

Increased protein synthesis during post-exercise period

unlikely to be due to increased transcription of RNA
Changes in protein synthesis independent of total RNA
more likely due to change in translational control of mRNA
Recent evidence points to involvement of translational
initiation factors (eIF4E & eIF4G)

Extent of post-exercise protein synthesis also

dependent on half-life of mRNA

Controlled by ribonucleases (degradative enzymes)

Other proteins stabilise and destabilise mRNA against
degradation by ribonucleases
48

Mitochondrial protein
synthesis

Mitochondria contain own

DNA and protein
synthesizing machinery
Mitochondrial genetic code
slightly different

Codon-anticodon interactions
simplified

Manage with only 22 species of

tRNA

Synthesis only small number

of proteins

Most mitochondrial proteins

coded for in nucleus and
transported into mitochondria

Adapted from: Tortora, GJ & Grabowski SR (2000) Principles of

Anatomy and Physiology (9th Ed). New York: John Wiley &
Sons. P84.

49

Proteins

Protein is a folded amino acid chain.

20 different amino acids can be found in
proteins.
A proteins 3-D structure determine its
functions in the cell.
Proteins do all essential work for the cells
build cellular structures
digest nutrients
execute metabolic functions
Mediate information flow within a cell
and among cellular communities.

Four levels of protein structure

Ribosomes

Polysomes

51

Post translational
modification
Folding

into a certain functional

structure
Mediated by another family of
protein: chaperon

52

Protein degradation
Protein

content of a cell depends

on balance between protein
synthesis and degradation

Change in protein = synthesis rate - degradation rate

53

Protein degradation

Three main protein degrading systems in muscle:

Ubiquitin-proteosome
Protein marked for degradation by attachment of ubiquitin
units
Inactive 20S proteosome activated by regulatory protein to
become active 26S proteosome
26S proteosome breaks protein into small peptides
Small peptides broken down into free amino acids by
other processes in cell

Lysosomal
Proteins enter lysosome via endocytosis
cathepsins and proteinases degrade bonds

Calpain
Calcium activated proteinase in cytosol of cell
Various isomers activated at different calcium
concentrations
54

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