PROTEIN SYNTHESIS

Uus Saepuloh
Biotechnology Laboratory
Primate Research Center Bogor Agricultural University
(PSSP LPPM IPB)

From Gene to Protein

GMO = Genetically Modified Organism

Featherless chicken
bovin somatotropin; used to increase
milk production
Extended shelf-life tomato

Herbicide resistant soybean

Golden Rice increased Vitamin A content

Principles of Biochemistry

Biochemistry is the application of chemistry to the study of

biological processes at the cellular and molecular level.
Cells (basic structural units of living organisms) are highly
organized and constant source of energy is required to
maintain the ordered state.
Living processes contain thousands of chemical pathways.
Precise regulation and integration of these pathways are
required to maintain life
All organisms use the same type of molecules:
carbohydrates, proteins, lipids & nucleic acids.
Instructions for growth, reproduction and developments for
each organism is encoded in their DNA

Nucleoid region contains the DNA

Cell membrane & cell wall
Contain ribosomes (no membrane)
to make proteins in
their cytoplasm

Contain 3 basic cell structures:

Nucleus
Cell Membrane
Cytoplasm with organelles

Prokaryotic and Eukaryotic Genome

Eukaryotic genome organization

Nucleosome

Histone

The Central Dogma

The Central Dogma describes the Flow of
Information from DNA
RNA Protein.

DNA Replication

Transcription

Translation

DNA Structure

DNA Replication
DNA Replication

DNA must replicate during each cell division

The Mechanism of DNA Replication

DNA replication is catalyzed by DNA polymerase
DNA polymerase needs an RNA primer
DNA polymerase adds nucleotides to the 3 end of the
growing strand
Nucleotides are added by complementary base pairing
with the template strand
The substrates, deoxyribonucleoside triphosphates, are
hydrolyzed as added, releasing energy for DNA
synthesis.

The Mechanism of DNA Replication

Many proteins assist in DNA replication
DNA helicases unwind the double helix, the
template strands are stabilized by other proteins
Single-stranded DNA binding proteins make the
template available

The Mechanism of DNA Replication

Many proteins assist in DNA replication

DNA helicases
Single-stranded DNA binding proteins
RNA primase catalyzes the synthesis of short
RNA primers, to which nucleotides are added.
DNA polymerase III extends the strand in the 5to-3 direction

RNA primase

The Mechanism of DNA Replication

DNA synthesis on the leading strand is continuous
The lagging strand grows the same general direction as
the leading strand (in the same direction as the
Replication Fork). However, DNA is made in the 5-to3 direction
Therefore, DNA synthesis on the lagging strand is
discontinuous
DNA is added as short fragments (Okasaki fragments)
that are subsequently ligated together

Transcription
DNA Replication

Transcription

Translation

A gene is expressed in two steps:

DNA is transcribed to RNA
Then RNA is translated into protein.

Figure 12.3

DNA and RNA differ

RNA differs from DNA in three ways:
RNA is single-stranded (but it can fold back
upon itself to form secondary structure, e.g.
tRNA)
In RNA, the sugar molecule is ribose rather
than deoxyribose
In RNA, the fourth base is uracil rather than
thymine.

DNA

RNA
H

OH

OH

OH

OH

DNA
RNA

RNA
U
OH

U
OH

OH

OH

OH

OH

OH

OH

OH

OH

 RNA is synthesized via a process

called Transcription
mRNA, rRNA and tRNA are
transcribed by similar mechanisms
Transcription has three phases:
Initiation
Elongation
Termination
RNA is transcribed from a DNA
template after the bases of DNA are
exposed by unwinding of the double
helix.
In a given region of DNA, only one of
the two strands can act as a
template for transcription.

Figure 12.4 Part 1

Transcription: Initiation

Unwind the DNA template: template and

complementary strands
Initiation: RNA polymerase recognizes and
binds to a promoter sequence on DNA

Figure 12.4 Part 1

Transcription: Elongation
Elongation: RNA polymerase elongates the nascent
RNA molecule in a 5-to-3 direction, antiparallel to
the template DNA
Nucleotides are added by complementary base pairing
with the template strand
The substrates, ribonucleoside triphosphates, are
hydrolyzed as added, releasing energy for RNA
synthesis.

Figure 12.4 Part 1

RNA processing enzymes recruited by the tail of

RNA polymerase II during the elongation process

Transcription: Termination
Termination: Special DNA sequences and
protein helpers terminate transcription.
The transcript is released from the DNA.
This Primary Transcript is called the premRNA
The pre-mRNA is processed to generate the
mature mRNA

Figure 12.4 Part 2

mRNA Transport

The Genetic Code

The genetic code consists of triplets of
nucleotides (codons)
Since there are four bases (A,C,G,T), there
are 64 possible codons (43):
1 start codon (encodes methionine)
3 termination codons (stop translation)
60 codons for the 20 amino acids

Hence, the genetic code is redundant; i.e.

there is more than one codon for certain
amino acids.
However, a single codon does not specify more
than one amino acid
The genetic code is universal (almost)

Figure 12.5

Translation
Translation if the process whereby mRNA is
decoded into protein
Prokaryotes: translation begins before mRNA
synthesis (transcription) is completed
Eukaryotes: transcription occurs in the nucleus
and translation occurs in the cytoplasm
Translation requires four components:
ribosomes, tRNAs, activating enzymes, and
mRNA (template)

Three types of RNA are involved in protein

synthesis
Messenger RNA [mRNA]
- The template
Ribosomal RNA [rRNA]

Transfer RNA [tRNA]

- adapter

 During translation, the order in which amino

acids are linked together is specified by the
codon in the mRNA
Transfer RNA (tRNA) acts as the adapter.
Via the anticodon-codon interaction, tRNA brings
the correct amino acid corresponding to the
mRNA codon.
Aminoacyl-tRNA synthetases, a family of
activating enzymes, attach specific amino acids
to their appropriate tRNAs, forming charged
tRNAs.

Transfer RNA The adapter

RNA is single-stranded but it can fold back
upon itself to form secondary structures

Figure 12.7

Ribosome
The mRNA meets the charged tRNAs at the
ribosome
The ribosome has two subunits: small and large
The ribosome has sites: E, P, A

Figure 12.9

Composition of the prokaryotic

and eukaryotic ribosome

Translation- Initiation
Translation has three phases:
Initiation
Elongation
Termination
Initiation: An initiation complex forms, consisting of
the initiator tRNA charged with methionine and the
small ribosomal subunit bound to mRNA triggers the
beginning of translation

Translation: initiation

Translation - Elongation
The ribosome moves along the mRNA one codon
at a time in a 5-to-3 direction
Polypeptides grow from the N terminus toward
the C terminus
Charged tRNAs bring amino acids to the
ribosome sequentially
Specificity is provided by:
the anticodon (tRNA) -codon (mRNA) interaction
the accuracy of aminoacyl-tRNA synthetases

Translation - Elongation

Figure 12.11 Part 1

Translation - Elongation

Figure 12.11 Part 2

Translation - Termination
The presence of a stop codon in the A site of
the ribosome causes translation to terminate
The completed protein is released

Translation
In a polysome,
more than one
ribosome
moves along
the mRNA at
one time

These proteins then move to their specific locations

in the cell to execute their specific functions

Post Translation Modification

Peptide chain undergoes folding

Some amino acids might be changed
Carbohydrates or lipids can be added
Peptide can be activated by addition or removal of
some residue (acetate, phosphate, methyl etc.)
Changes in the Hydrogen bond proclivity which
results in secondary and tertiary structures
Some of the proteins might remain in cytosol while
others are transported across the membrane or even
imported into cellular organelles (mitochondria or
chloroplasts) to accomplish their functions

Types of Post-translational modifications

Proteolytic cleavage
Glycosylation
Methylation
Hydroxylation
Phosphorylation
Sulfation
Acylation
Carboxylation
Formylation
Disulfide bond
formation