The Genetic Code

&
Translation Process
(Protein Biosynthesis)

Department of Biochemistry and Molecular Biology

School of Medicine, Tarumanagara University
October 2017
 The Genetic Code
• The genetic code is the set of rules by which information encoded in genetic
material (DNA or RNA sequences) is translated into proteins (amino acid
sequences) by living cells.

• mRNA molecules have no affinity for amino acids and, therefore, that the translation
of the information in the mRNA nucleotide sequence into the amino acid sequence of
a protein requires an intermediate adapter molecule.

• This adapter molecule (tRNA) must recognize a specific nucleotide sequence on the
one hand as well as a specific amino acid on the other.

• Twenty amino acids are required for the synthesis of the cellular proteins; thus, there
must be at least 20 distinct codons that make up the genetic code.

• Since there are only 4 different nucleotides in mRNA, each codon must consist of
more than a single purine or pyrimidine nucleotide. Codons consisting of two
nucleotides each could provide for only 16 (42) specific codons, whereas codons
of three nucleotides could provide 64 (43) specific codons.
• The mRNA transcript is a linear sequence of
nucleotides carrying genetic information and it is
single-stranded.

• Every three bases of mRNA (a triplet) specifies an

amino acid to be added to a growing polypeptide
chain; the relationship between the triplets and
the corresponding amino acids is the genetic code.

• Each base triplet of mRNA is called a codon. The

genetic code is nearly universal for all forms of life.
There are intriguing exception of a few minor variations in mitochondria, some bacteria, and some single-
celled eukaryotes
 The Genetic Code
stop codons: 3 codons (UUA, UGA, UAG) in the
genetic code used to terminate translation
start codon: the codon (AUG) used to signify the
start of translation
In bacteria : AUG, GUG, or UUG (5’-3’)
The remainder of the code is degenerate
meaning that some amino acids are specified
by more than one codon.

10
Features of the Genetic Code
2. Wobble allows some tRNAs to recognize more than one codon
When several different codons specify one
amino acid, the difference between them
usually lies at the third base position (at the 3’
end).
-The first two letters of each codon are the
primary determinants of specificity, a feature
that has some interesting consequences.

Anticodon
-Transfer RNAs base-pair with mRNA
codons at a three-base sequence on the
tRNA.
-The third base of most codons pairs rather
loosely with the corresponding base of its
anticodon; to use his picturesque word, the
third base of such codons (and the first base
of their corresponding anticodons) “wobbles.”
-Crick proposed the Wobble hypothesis:

FIGURE 27-8a Pairing relationship of codon and anticodon.

(a) Alignment of the two RNAs is antiparallel. The tRNA is
shown in the traditional cloverleaf configuration.
MARSHALL Nirenberg (1961)

Relationship between mRNA and the Protein Product

• The order of the codons in the mRNA determines the sequence in which
amino acids are added to the growing polypeptida chain.

• The order of the codons in the mRNA determines the linear sequence of
amino acids in the protein.

TYPE OF MUTATIONS
TYPE Description Example

Point > A Single base change

Silent- > A change that specifies the same amino CGA .-> CGG
Arg->Arg
Missense- > Different Amino CGA->CCA
Arg -> Pro
Nonsense - >Produce stop kodon CGA->UGA
Arg > Stop
Insertion- > Addition one or more bases
Deletion- > Loss one or more bases
 Translation of mRNA
(Protein Synthesis)

• Translation is the process by which the nucleotide sequence of

mRNA is converveted to the amino acid sequence of a polypeptide.

• In the first step of the process, all the components needed for
translation come together.
 Protein Synthesis

1. Protein biosynthesis takes place in five stages

2. The ribosome is a complex supramolecular machine

3. ```Transfer RNAs have characteristic structural features

4. Stage 1: Amionoacyl-tRNA synthetases attach the correct amino acids to their tRNAs

5. Stage 2: A specific amino acid initiates protein synthesis

6. Stage 3: Peptide bonds are formed in the elongation stage

7. Stage 4: Termination of polypeptide synthesis requires a special signal

8. Stage 5: Newly synthesized polypeptide chains undergo folding and processing

9. Protein synthesis is inhibited by many antibiotics and toxins

 2. Protein Synthesis
1. Protein biosynthesis takes place in five stages
2. The ribosome is a complex supramolecular machine
3. Transfer RNAs have characteristic structural features
4. Stage 1: Amionoacyl-tRNA synthetases attach the correct amino acids to their tRNAs
5. Stage 2: A specific amino acid initiates protein synthesis
6. Stage 3: Peptide bonds are formed in the elongation stage
7. Stage 4: Termination of polypeptide synthesis requires a special signal
8. Stage 5: Newly synthesized polypeptide chains undergo folding and processing
9. Protein synthesis is inhibited by many antibiotics and toxins
mRNA
 Prokaryotic mRNA (polycistrionic)

2019/11/20 23
Eukaryotic mRNA (monocistrionic)
(Kozak Scanning)
Formation of aminoacyl-tRNA
Amino acid activation

aminoacyl-tRNA synthetase
Translation initiation pathway
in E. coli ribosom
Translational initiation
pathway in eukaryotes.
Initiation factors are represented
by colored rectangles where they
are first implicated in the pathway
and by circles of the same color
thereafter. .

Several of the initiation factors:

(4A, 4B, 4E, 4G, and 4H)
have been left out of the
48S initiation complex for clarity
Recognition of the codon by the anticodon
A comparison of the structures of procaryotic and eucaryotic ribosomes
Translational initiation
pathway in eukaryotes.
Initiation factors are represented
by colored rectangles where they
are first implicated in the pathway
and by circles of the same color
thereafter. .

Several of the initiation factors:

(4A, 4B, 4E, 4G, and 4H)
have been left out of the
48S initiation complex for clarity
• The flow of genetic information follows the sequence DNA → RNA → protein.
• The genetic information in the structural region of a gene is transcribed into an RNA
molecule such that the sequence of the latter is complementary to that in the DNA.

• Ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), are
directly involved in protein synthesis; miRNAs regulate mRNA function at the level
of translation and/or stability.

• The information in mRNA is in a tandem array of codons, each of which is 3 nt long.

• mRNA is read continuously from a start codon (AUG) to a termination codon

• The open reading frame, or ORF, of the mRNA is the series of codons, each specifying
a certain amino acid, that determines the precise aa sequence of the protein.

• Protein synthesis, like DNA and RNA synthesis, follows the 5' to 3' polarity of mRNA
and can be divided into three processes: initiation, elongation, and termination.

• Mutant proteins arise when single-base substitutions result in codons that specify a
different amino acid at a given position, when a stop codon results in a truncated protein,
or when base additions or deletions alter the reading frame, so different codons are read.

• A variety of compounds, including several antibiotics, inhibit protein synthesis by

affecting one or more of the steps involved in protein synthesis.
Inhibitors of Protein or RNA Synthesis
Acting Only on Procaryotes*
• Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome
• Streptomycin prevents the transition from initiation complex to chain-elongating
ribosome and also causes miscoding.
• Chloramphenicol blocks the peptidyl transferase reaction on ribosomes
• Erythromycin blocks the translocation reaction on ribosomes
• Rifamycin blocks initiation of RNA chains by binding to RNA polymerase (prevents
RNA synthesis)

Acting Only on Procaryotes and Eucaryotes

• Puromycin causes the premature release of nascent polypeptide chains by its
addition to growing chain end.
• Actinomycin D binds to DNA and blocks the movement of RNA polymerase
(prevents RNA synthesis)

Acting Only on Eucaryotes

• Cycloheximide blocks the translocation reaction on ribosomes.

• Anisomycin blocks the peptidyl transferase reaction on ribosomes
• α-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II
 POST-TRANSLATIONAL MODIFICATION

 To become mature proteins, polypeptides must fold to

their native conformations, their disulfide bonds, if any,
must form, and, in the case of multisubunit proteins, the
subunits must properly combine.

 Moreover, as we have seen throughout this text, many proteins

are modified in enzymatic reactions that proteolytically cleave
certain peptide bonds and/or derivatize specific residues.

A. Proteolytic Cleavage
Proteolytic removal of their leading Met (or fMet) residue
shortly after it emerges from the ribosome.

Signal Peptides Are Removed from Nascent Proteins by a Signal Peptidase

B. Covalent Modification

These include acetylations, glycosylations, hydroxylations, methylations,

nucleotidylations, phosphorylations, and ADP-ribosylations as well as numerous
“miscellaneous” modifications.

Collagen Assembly Requires Chemical Modification: OH-Lys & OH-Pro

C. Folding Accessory Proteins

Levinthal Paradox: 32n ~ 10n

For protein with 100 amino acids : t = 1087

1. Molecular Chaperones: The GroEL/ES System

2. Protein Disulfide Isomerase
3. Peptidyl Prolyl Cis–Trans Isomerase
Polypeptide Synthesis: An Overview
a. Polypeptide Synthesis Proceeds from N-Terminus to C-Terminus
(Howard Dintzis, (1961)
b. Ribosomes Read mRNA in the 5’ → 3’ direction

C. Active Translation Occurs on Polyribosomes

d. Chain Elongation Occurs by the Linkage of the Growing Polypeptide
to the Incoming tRNA’s Amino Acid Residue

Chain Initiation
a. fMet Is the N-Terminal Residue of Prokaryotic Polypeptides
b. Base Pairing between mRNA and the 16S rRNA Helps Select the
Translational Initiation Site
c. Prokaryotic Initiation Is a Three-Stage Process that Requires the
Participation of Soluble Protein Initiation Factors
d. Eukaryotic Initiation Is Far More Complicated than That of Prokaryotes
POSTTRANSLATIONAL MODIFICATION

A. Proteolytic Cleavage
B. Signal Peptides Are Removed from Nascent Proteins by a Signal Peptidase
C. Polyproteins
D. Covalent Modification:
- Collagen Assembly Requires Chemical Modification
Additional PTMs
• Nitrosylation
• Glycation
• ADP-ribosylation
• O-GlcNAc
• Lipidation, ex:- Glycophosphatidyl inositol
(GPI) membrane protein
- Prenyl groups,
Farnesylation
Protein Folding

• Protein disulfide isomerase (PDI)

• Molecular Chaperones:

• heat shock proteins (Hsp), ex: HSP70

• Chaperonins:
• Hsp60 proteins (GroEL in E. coli)
• Hsp10 proteins (GroES in E. coli),

The X-ray structure of the GroEL–GroES–(ADP)7 chaperonin complex

 Hsp70 in de novo protein biogenesis

 Hsp70 is believed to bind and stabilize nascent polypeptides early in their

synthesis--preventing misfolding and aggregation
 Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to
the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for
folding (e.g., chaperonins)
 Hsp70 is also important during cellular stresses (thermotolerance), and has
numerous other functions in the cell apart from assisting de novo protein folding. It
often works in collaboration with other chaperones, especially Hsp40
REGULASI EKSPRESI GEN

• Ekspresi gen merupakan keseluruhan proses, di mana

informasi genetik yang disimpan dalam DNA diubah
menjadi produk fungsional gen yaitu RNA atau protein.

• Tujuan regulasi ekspresi gen adalah agar ekspresinya

terjamin secara akurat baik spatial maupun temporal,
sehingga di satu pihak sel dapat beradaptasi terhadap
perubahan lingkungan, sedangkan di pihak lain sel dapat
tumbuh dan berdiferensiasi secara normal.

• Untuk mencapai tujuan tersebut , regulasi ekspresi gen

terjadi dalam berbagai tingkat.
 REGULASI EXPRESSI GENE
(1) controlling when and how often a given geneis transcribed (transcriptional control),
(2) controlling how the primary RNA transcript is spliced or otherwise processed
(RNA processing control),
(3) selecting which completed mRNAs in the cell nucleus are exported to the cytoplasm
(RNA transport control),
(4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5)
selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control) (6)
selectively activating, inactivating, or compartmentalizing specific protein molecules after they
have been made (protein activity control)
Levels of regulation
of eucaryotic gene
expression.
• The flow of genetic information follows the sequence DNA → RNA → protein.
• The genetic information in the structural region of a gene is transcribed into an RNA
molecule such that the sequence of the latter is complementary to that in the DNA.

• Ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), are
directly involved in protein synthesis; miRNAs regulate mRNA function at the level
of translation and/or stability.

• The information in mRNA is in a tandem array of codons, each of which is 3 nt long.

• mRNA is read continuously from a start codon (AUG) to a termination codon

• The open reading frame, or ORF, of the mRNA is the series of codons, each specifying
a certain amino acid, that determines the precise aa sequence of the protein.

• Protein synthesis, like DNA and RNA synthesis, follows the 5' to 3' polarity of mRNA
and can be divided into three processes: initiation, elongation, and termination.

• Mutant proteins arise when single-base substitutions result in codons that specify a
different amino acid at a given position, when a stop codon results in a truncated protein,
or when base additions or deletions alter the reading frame, so different codons are read.

• A variety of compounds, including several antibiotics, inhibit protein synthesis by

affecting one or more of the steps involved in protein synthesis.
Structure of the Proteasome