Gene expression & Protein

synthesis

Abdul Salam M. Sofro

Faculty of Medicine
YARSI University Jakarta

Anak laki-laki bernama R umur 6 th,

jatuh dari belajar sepeda, lutut kanan
memar sedikit. Makin hari makin
bengkak dan membiru. Oleh bapaknya,
R dibawa ke RSUD. Oleh dokter yang
memeriksa, dikatakan bahwa R
menderita Hemofilia dan harus dirawat
di RS. Selama perawatan di RS, R diberi
transfusi Faktor VIII penggumpalan
darah.

Learning objectives
By the end of the lecture:
Students are expected to
understand the principles of gene
expression
students are expected to
understand and able to describe
the molecular or biochemical
processes underlying protein
synthesis

Core topics
Overview of Gene expression
Transcription of DNA (RNA
synthesis)
Protein synthesis (translation)

Overview of gene
expression

Central dogma of Life

www.austincc.edu/.../1308ProteinSynthesis.pp

Transfer of genetic information

Replication of DNA
transmission of genetic information
from parental cell to its daughter cells
Transcription of DNA
transmission of genetic information
from DNA to RNA
Translation of RNA
(polypeptide/protein biosynthesis)
transmission of genetic information
from RNA to polypeptide/protein

 Protein biosynthesis is also called

translation (translation of information from
four-letter language & structure of nucleic
acid into 20-letter language & structure of
protein)
This process requires:
Informational mRNA exported from
nucleus
bilingual tRNA that reads the message
Ribosomes that serve as catalytic &
organizational centers
A variety of protein factors & energy

Cont.
Polypeptide/proteins are formed by
sequential addition of amino acids in
the specific order determined by info
carried in the nucleotide sequence of
the mRNA
Proteins are often matured or
processed by a variety of
modifications
Levels of translation is regulated

 Cells vary in their need & ability to

synthesized proteins:
Growing cells & dividing cells must
synthesize much larger amounts of
protein
Some cells synthesize proteins for
export as well as for their own use
(e.g. liver cells synthesize large
numbers of enzymes needed for
their metabolic pathways as well as
proteins for export including serum
albumin)

 Terminally differentiated (adult) red

blood cells have no nuclei, do not
divide & do not synthesize proteins
due to the absence of components
of the biosynthetic apparatus

Transcription of DNA
(RNA synthesis)

 Transcription produces components of

the translational apparatus
mRNA
rRNA of Ribosomes
tRNA

RNA (ribonucleic acid)

Sugar is ribose
RNA is a polymer of
ribonucleotides.
Bases are adenine, guanine,
cytosine and uracil (instead of
thymine)
Single strand
Three types of RNA: mRNA, tRNA
& rRNA

mRNA
Carrier of information present in DNA
In eukaryotes (including human)
usually are synthesized as larger
precursor molecules that are
processed prior to export from the
nucleus
almost always monocistronic
(encoding a single polypeptide)

rRNA
Together with ribosomal proteins
constitute ribosome which in turn
serve as workbenches for
polypeptide/protein biosynthesis
Ribosome have two dissimilar
subunits (small & large subunits),
each contains RNA & many proteins

tRNA
A bilingual translator molecule
All tRNA molecules have several
common structural characteristics
(3-terminal CCA sequence to bind
amino acid, a highly conserved
cloverleaf secondary structure & Lshape three dimensional structure)
Great specificity in interaction with
mRNA & the aminoacyl-tRNA
synthetase

Transcription of DNA (RNA

synthesis)
In chromosomes, DNA acts as a
template for the synthesis of RNA in a
process called transcription:
Only one strand of DNA act as
template (35 strand)
Originated from any point of DNA of
the gene (Polypeptide gene, tRNA
gene or rRNA gene) at the promotor
site
Does not require RNA primer

 Involved:
RNA polymerase
NTP (ATP, GTP, CTP, UTP)
Termination signal
In most mammalian cells, only 1% of
the DNA sequence is copied into a
functional RNA (mRNA). Only one part
of the DNA is transcribed to produce
nuclear RNA, and only a minor portion
of the nuclear RNA survives the RNA
processing steps.

Trancription of RNA from DNA (note: DNA antisense is

used as the template

Promoter
Bind RNA polymerase protect DNA
from digestion
Two common motifs on 5 : -10
sequence 5-TATAAT-3 and -35
sequence (6 bp long) 5-TTGACA-3
At coding strand = sense (+) strand
& template strand = antisense (-)
strand

 Strong vs. weak promoter (every 2

sec & once in 10 min.)
Specific sequences near it
influenced by regulatory proteins &
interact with RNA polymerase
Recognized by sigma subunit RNA
polymerase (2 holoenzyme)

http://www.tokresource.org/tok_classes/biobiobio/biomenu/transcription_

RNA polymerase
Searches DNA for initiation site
There are many more molecules of RNA
polymerase per cell than DNA
polymerase.
RNA polymerase proceeds at a rate
much slower than DNA polymerase
(approximately 50-100 bases/sec for
RNA versus near 1000 bases/sec for
DNA
the fidelity of RNA polymerization is

 Unwinds a short stretch of double-helical

DNA to produce DNA template
Select correct dNTP & catalyses formation
of fosfodiester bond
Interact with activator & repressor protein
that modulate the rate of transcription
Unwinds nearly two turns of template DNA
before initiating RNA synthesis
Starts with pppG or pppA
Primers are not needed

DNA template
Transcription bubble for elongation
containing RNA pol, DNA, nascent
RNA
Form RNA-DNA hybrid helix (about 12
bp long/one turn of A-DNA)
Direct RNA synthesis
Transcribed by RNA pol (lack
nuclease activity) with lower fidelity
than that of replication (error rate 1
in 104 or 105)

Transcription termination

Formation of fosfodiester bonds ceases

RNA-DNA hybrid dissociates
Melted DNA region rewinds
RNA pol releases DNA
Precisely controlled
Stop signals in DNA template regions
e.g. palindromic GC-rich region
followed by AT-rich region forms
RNA hairpin structure
Rho protein helps terminate
transcription

Nascent RNA & processing

Undergo little or no modification for mRNA
(maybe translated while being transcribed)
Cleaved & modified for rRNA & tRNA in E
coli, a primary RNA transcript is excised to
generate three rRNAs (5S, 16S & 23S) &
one tRNA by ribonuclease P
May contain arrays of several kinds of
tRNAs or several copies of same tRNA
Addition of nucleotides to termini of some
RNA chains (CCA to 3 tRNA)
Modifications of bases & ribose units of
rRNAs

 Processing of mRNA:
Capping at 5-end
Polyadenylation (tailing) at 3-end
Splicing removal of introns transcript
leaving exons transcript

One of the most

important stages in
RNA processing is
RNA splicing. In
many genes, the
DNA sequence
coding for proteins,
or "exons", may be
interrupted by
stretches of noncoding DNA, called
"introns".

In the cell nucleus, the DNA that

includes all the exons and introns
of the gene is first transcribed into
a complementary RNA copy called
"nuclear RNA," or nRNA.

In a second step, introns are

removed from nRNA by a process
called RNA splicing. The edited
sequence is called "messenger
RNA," or mRNA.

http://vizonline.visitationacademy.org/upperSchool/essig/A
_gen.htm

Protein synthesis
(Translation of mRNA)

In eukaryotic cells,
translation is carried
out within cytoplasm

http://kentsimmons.uwinnipeg.
ca/cm1504/proteinsynth.htm

Translation of RNA
The ribosome binds to the mRNA at
the start codon (AUG) that is
recognized only by the initiator tRNA.
The ribosome proceeds to the
elongation phase of protein
synthesis. During this stage,
complexes, composed of an amino
acid linked to tRNA, sequentially bind
to the appropriate codon in mRNA by
forming complementary base pairs
with the tRNA anticodon.

 The ribosome moves from codon to

codon along the mRNA. Amino acids
are added one by one, translated into
polypeptidic sequences dictated by
DNA and represented by mRNA. At
the end, a release factor binds to the
stop codon, terminating translation
and releasing the complete
polypeptide from the ribosome.

tRNA molecule

http://kentsimmons.uwinnipeg.ca/cm1504/proteinsynth
.htm

Activation of amino
acid

Codon
Three-letter code words ( a triplet
code)
Unambiguous
Non-overlapping
Without punctuation
Universal
Can be found either in DNA (sense
strand) and mRNA
The collection of codons (64) makes up the genetic code

The Genetic Code

5'
U

U
UUU
UUC
UUA
UUG

Phe
Phe
Leu
Leu

C
UCU
UCC
UCA
UCG

CUU
CUC
CUA
CUG

Leu
Leu
Leu
Leu

CCU
CCC
CCA
CCG

Pro
Pro
Pro
Pro

CAU
CAC
CAA
CAG

Tyr
Tyr
Stop
Stop
His
His
Gln
Gln

Ile
Ile
Ile
Met*
Val
Val
Val
Val

ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG

Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala

AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG

Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu

AUU
A AUC
AUA
AUG
GUU
G GUC
GUA
GUG

A
Ser
Ser
Ser
Ser

UAU
UAC
UAA
UAG

G
UGU
UGC
UGA
UGG

3'
Cys
Cys
Stop
Trp

U
C
A
G

CGU
CGC
CGA
CGG

Arg
Arg
Arg
Arg

U
C
A
G

AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG

Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly

U
C
A
G
U
C
A
G

*AUG signals translation initiation as well as coding for Met

Three
nonsense
codons (UAA,
UAG, UGA) do
not code for
specific amino
acid and are
utilized as
termination
signal

 Three of these possible codons

specify the termination of the
polypeptide chain. They are called
"stop codons (nonsense codons).
That leaves 61 codons to specify
only 20 different amino acids.
Therefore, most of the amino acids
are represented by more than one
codon. The genetic code is said
to be degenerate.

Amino acids specified by each codon sequence

on mRNA
Ala: Alanine

Cys:
Cysteine

Phe:
Phenylalanine Gly: Glycine

Asp: Aspartic Glu: Glutamic

acid
acid
Ile:
His: Histidine
Isoleucine

Lys: Lysine

Met:
Leu: Leucine
Methionine

Asn:
Asparagine

Pro: Proline

Gln:
Glutamine

Arg: Arginine

Ser: Serine

Thr:
Threonine

Val: Valine

Trp:
Tryptophane

Tyr: Tyrosisne

Protein translation takes place by the

following steps
1. Formation of the initiation complex
2. Elongation of the polypeptide chain
(one repetition of the steps a, b and c
for every amino acid incorporated into
the protein being made):
a. binding of aminoacyl-tRNA
b. peptide bond formation
c. translocation
3. Termination

Translation step 1: initiation

small ribosomal subunit and first tRNA meet

at the start codon AUG
joined by the large ribosomal subunit

Initiation of Polypeptide Chain

In eukaryotes consists of
three stages
forming the pre-initiation
complex
migration of mRNA
forming the full ribosomal
complex

Translation step 2: elongation

Polypeptide Elongation

Elongation

Translation step 3: termination

ends when the stop codon is

reached
stop codon is recognised by release

Initiation

Elongation

Termination

Mutations

Mutations
Result when changes occur in the
nucleotide sequence may not
occur in the template strand but
appear after replication
Some mutations occur by base
substitution single base changes
(point mutations):
Transitions (pryrimidine to other
pyrimidine, purine to other purine)
Transversion (pyrimidine to purine
or purine to pyrimidine)

 Single base changes in DNA

sequence followed by changes in
mRNA molecules may have one of
several effects when translated into
protein:
No detectable effect silent
mutation
Missense effect missense
mutation
Appearance of nonsense codon
that result in premature

 Substitution of amino acids in protein

causes missense mutations (illustration
on Hemoglobin molecule):
Acceptable missense mutations
Hb Hikari: AAA or AAG (lys) to AAU or
AAC (asp)
Hb E: GAA or GAG (glu) to AAA or AAG
(lys)
Partially acceptable missense mutations
Hb S: GAA or GAG (glu) to GUA or GUG
(val)
Unacceptable missense mutations
2+

3+

 Frameshift mutations result from

deletion or insertion of nucleotides
generates altered mRNAs
May be one, two, three or multiples
nucleotides

Inhibition of protein
synthesis

Some drugs are designed to directly

killing microorganisms (bactericidal) or
by inhibiting their growth
(bacteriostatic).

Some of them are based

on the inhibition of protein
synthesis

http://classes.midlandstech.com/carterp/Courses/bio225/chap
20/ss2.htm

http://classes.midlandstech.com/carterp/Courses/bio225/chap
20/ss2.htm