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 5’to-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

3

U

OH

2

OH

OH

OH

1

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, tRNA’s, 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 tRNA’s, forming charged
tRNA’s.

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 tRNA’s 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 tRNA’s 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