PROTEIN SYNTHESIS &

REGULATION OF GENE
EXPRESSION
Abdul Salam M. Sofro
Faculty of Medicine & LPPM YARSI
University Jakarta
RC Biotechnology UGM Yogyakarta

Teaching aims
By the end of the lecture:
students are expected to understand
the molecular or biochemical processes
of DNA replication, transcription and
protein synthesis
Students are expected to understand
the principles of gene expression

Core topics

Overview
DNA Structure & replication of DNA
Transcription of DNA (RNA synthesis)
Protein synthesis (translation)
Regulation of gene expression

Overview
Protein biosynthesis is also called translation
(translation of information from four-letter
language & structure of nucleic acid into 20letter 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

 Components of the translational

apparatus
mRNA
Ribosomes
tRNA

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
It has several identifying
characteristics:
almost always monocistronic (encoding
a single polypeptide)

 5 end is capped with 7-methylG

followed by non-translated region which
may be short or up to a few hundred
nucleotides in length separated the cap
with translational initiation signal (AUG)
Uninterrupted sequences that specify a
unique polypeptide sequence; followed
by 3-untranslated sequence, usually
about 100 nucleotides in length, before
terminated by a 100-200 nucleotide long
poly-A tail

 In prokaryotes:
5 terminus is not capped
Mostly polycistronic (encoding several
polypeptides & include more than one
initiation AUG sequence)
Ribosome positioning sequence is
located about 10 nucleotides upstream
of a valid AUG initiation signal
An untranslated sequence follows the
termination signal, but no poly-A tail

Ribosome
Workbenches for polypeptide/protein
biosynthesis
Have two dissimilar 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 &
L-shape three dimensional structure)
Great specificity in interaction with mRNA &
the aminoacyl-tRNA synthetase

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

DNA structure & Replication of

DNA

DNA structure & Replication of DNA

DNA is a macromolecule that ultimately
controls every aspect of cellular
functions through protein synthesis
DNA is a transforming agent as well as
material responsible for transmitting
genetic information from one generation
to the next
DNA

RNA

Protein

Human Genome Size

NUCLEAR GENOME
* 23 pairs of chromosomes 2
x ( 3 x 109 b.p) 2 meters
DNA / Cell
* 2 x ( 3 x 1012 cells)
meters DNA in human body
8,000 x earth to moon
MITOCHONDRIAL GENOME
* circular, 16,569 bp

DNA (deoxyribonucleic acid)

Sugar is deoxyribose
DNA is a polymer of
deoxyribonucleotides
Bases are adenine (A), guanine (G),
cytosine (C) and thymine (T)
Double strands anti parallel

Replication of DNA
Takes place in nucleus
Both strands act as template (35 strand)
Originated from replication fork or
replication bubble
Factors involved:
Helicase
DNA binding proteins
DNA polymerase
Primase
dNTP (dATP, dGTP, dCTP, TTP) & many
others

 Needs RNA primer

Produces :
Leading strand of new DNA
(complementary to old DNA template
with free 3-OH end)
Lagging strand of new DNA with
Okazaki fragments (complementary to
old DNA template with free 5- end

Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html

Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html

Transcription of DNA
(RNA synthesis)

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

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.

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)

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 much
lower than DNA

 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)

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

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

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.

Protein synthesis
(Translation of mRNA)

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.

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

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

A = adenine G = guanine C = cytosine

T = thymine U = uracil
DNA transfers information to mRNA in the
form of a code defined by a sequence of
nucleotides bases.
During protein synthesis, ribosomes move
along the mRNA molecule and "read" its
sequence three nucleotides at a time
(codon) from the 5' end to the 3' end.

 Each amino acid is specified by the

mRNA's codon, and then pairs with a
sequence of three complementary
nucleotides carried by a particular tRNA
(anticodon).
Since RNA is constructed from four
types of nucleotides, there are 64
possible triplet sequences or codons
(4x4x4).

 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

Remember !
Proteins are polypeptides made up of
individual amino acids linked together,
Carbohydrates are polysaccharides
made up of individual monosaccharides
linked together, and
Nucleic acids are polynucleotides made
up of individual nucleotides linked
together.

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 termination of polypeptide
chain being synthesized nonsense
mutation

 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
Hb M: Hb (Fe2+) to met Hb (Fe3+)

 Frameshift mutations result from

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

Regulation of gene expression

 In bacteria & viruses:

Alteration of gene expression is
required by organism to adapt to
environmental changes involves
interaction of specific binding proteins
with various regions of DNA in the
immediate vicinity of the transcription
start site

 In eukaryotes:
in addition to those proteins,
alteration of gene expression also
involves tissue specific expression;
regulation by hormones, metals &
chemicals; gene amplification; gene
rearrangement; posttranscriptional
modification

Type of responses to a regulatory signal

Type A: increased rate of gene
expression that is dependent upon the
continued presence of inducing signal
Type B: increased rate of gene
expression that transient even in the
continued present of regulatory signal
Type C: increased rate of gene
expression that persist indefinitely even
after the termination of the signal

Type of gene regulation

Positive regulation:
The expression of genetic info is
quantitatively increased by the presence
of a specific regulatory element (the
molecule is positive regulator)
Negative regulation:
the expression of genetic info is
diminished by the presence of a specific
regulatory element (the molecule is
negative regulator)

Model of gene expression in prokaryotes

One cistron, one subunit concept instead
of one gene one enzyme concept (cistron
is the smallest unit of gene expression,
coding for the structure of the subunit
of a protein molecule)
Inducible genes: their expression
increases in response to an inducer
Constitutive genes: their expression is
reasonably constant (not known to be
subject to regulation)

The earliest level of regulation is at DNA level during transcription

Legend:
Process of creating a hybrid strand of DNA/RNA
The two strands of a DNA molecule are denatured by heating to about
100C = 212F (a to b). At this temperature, the complementary base pairs
that hold the double helix strands together are disrupted and the helix
rapidly dissociates into two single strands.
The DNA denaturation is reversible by keeping the two single stands of
DNA for a prolonged period at 65C = 149F (b to a). This process is called
DNA renaturation or hybridization.
Similar hybridization reactions can occur between any single stranded
nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript
is introduced during the renaturation process, the RNA competes with the
coding DNA strand and forms double-stranded DNA/RNA hybrid molecule
(c to d).
These hybridization reactions can be used to detect and characterize
nucleotide sequences using a particular nucleotide sequence as a probe.