mRNA processing, genetic

code, and protein synthesis

CP 10.1: menjelaskan pemroses mRNA

CP 10.2: menjelaskan kode genetik

CP 10.3: menjelaskan aspek-aspek sintesis protein

CP 10.4: menjelaskan meknisme sintesis protein

mRNA PROCESSING
 Processing of mRNA
There is essentially no processing of prokaryotic mRNA; it
can start to be translated before it has finished being
transcribed.
In eukaryotes, mRNA is synthesized by RNA Pol II as longer
precursors (pre-mRNA), the population of different pre-
mRNAs being called heterogeneous nuclear RNA (hnRNA).
Specific proteins bind to hnRNA to form hnRNP and then
small nuclear RNP (snRNP) particles interact with hnRNP to
carry out some of the RNA processing events.
Processing of eukaryotic hnRNA involves four events: 5-
capping, 3-cleavage and polyadenylation, splicing and
methylation.

CP 10.1: menjelaskan pemroses mRNA

 hnRNP
RNA Pol II transcripts (hnRNA) complex with the three most
abundant hnRNP proteins, the A, B and C proteins, to form
hnRNP particles.
These contain three copies of three tetramers and around
600700 nucleotides of hnRNA.
They assist RNA processing events.

CP 10.1: menjelaskan pemroses mRNA

 snRNP particles
There are many uracil-rich snRNA molecules made by RNA
Pol II which complex with specific proteins to form snRNPs.
The most abundant are involved in splicing, and a large
number define methylation sites in pre-rRNA.
Those containing the sequence 5-RA(U)nGR-3 bind eight
common proteins in the cytoplasm, become
hypermethylated and are imported back into the nucleus.

CP 10.1: menjelaskan pemroses mRNA

 5 Capping
This is the addition of a 7-
methylguanosine nucleotide
(m7G) to the 5-end of an
RNA Pol II transcript when it
is about 25 nt long.
The m7G, or cap, is added
in reverse polarity (5 to 5),
thus acting as a barrier to 5-
exonuclease attack, but it
also promotes splicing,
transport and translation.

CP 10.1: menjelaskan pemroses mRNA

 3 Cleavage and polyadenylation
Most eukaryotic pre-mRNAs are cleaved at a
polyadenylation site and poly(A) polymerase (PAP) then
adds a poly(A) tail of around 250 nt to generate the mature
3-end.

CP 10.1: menjelaskan pemroses mRNA

 Splicing
In eukaryotic pre-mRNA processing, intervening sequences
(introns) that interrupt the coding regions (exons) are
removed (spliced out), and the two flanking exons are
joined.
This splicing reaction occurs in the nucleus and requires the
intron to have a 5-GU, an AG-3 and a branchpoint
sequence.
In a two-step reaction, the intron is removed as a tailed
circular molecule, or lariat, and is degraded.
Splicing involves the binding of snRNPs to the conserved
sequences to form a spliceosome in which the cleavage
and ligation reactions take place.

CP 10.1: menjelaskan pemroses mRNA

 Splicing

CP 10.1: menjelaskan pemroses mRNA

 THE GENETIC CODE
Nature
The genetic code is the way in which the nucleotide
sequence in nucleic acids specifies the amino acid
sequence in proteins.
It is a triplet code, where the codons (groups of three
nucleotides) are adjacent (nonoverlapping) and are not
separated by punctuation (comma-less).
Because many of the 64 codons specify the same amino
acid, the genetic code is degenerate (has redundancy).

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
Deciphering
The standard genetic code was deciphered by adding homopolymers,
copolymers or synthetic nucleotide triplets to cell extracts which were
capable of limited translation.
It was found that 61 codons specify the 20 amino acids and there are
three stop codons.

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
Features
Eighteen of the 20 amino acids are specified by multiple (or
synonymous) codons which are grouped together in the
genetic code table.
Usually they differ only in the third codon position.
If this is a pyrimidine, then the codons always specify the
same amino acid. If a purine, then this is usually also true.

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
Effect of mutation
The grouping of synonymous codons means that the effects
of mutations are minimized.
Transitions in the third position often have no effect, as do
transversions more than half the time.
Mutations in the first and second position often result in a
chemically similar type of amino acid being used.

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
Universality
Until recently, the standard genetic code was considered
universal: however, some deviations are now known to
occur in mitochondria and some unicellular organisms.

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
ORFs
Open reading frames are suspected coding regions usually
identified by computer in newly sequenced DNA.
They are continuous groups of adjacent codons following a
start codon and ending at a stop codon.

CP 10.2: menjelaskan kode genetik

 THE GENETIC CODE
Overlapping genes
These occur when the coding region of one gene partially or
completely overlaps that of another.
Thus one reading frame encodes one protein, and one of
the other possible frames encodes part or all of a second
protein.
Some small viral genomes use this strategy to increase the
coding capacity of their genomes.

CP 10.2: menjelaskan kode genetik

 ASPECTS OF PROTEIN SYNTHESIS
Codonanticodon interaction
In the cleft of the
ribosome, an antiparallel
formation of three base
pairs occurs between the
codon on the mRNA and
the anticodon on the
tRNA.
If the 5 anticodon base is
modified, the tRNA can
usually interact with more
than one codon.

CP 10.3: menjelaskan aspek-aspek sintesis protein

 ASPECTS OF PROTEIN SYNTHESIS
Wobble
The wobble hypothesis describes the nonstandard base
pairs that can form between modified 5-anticodon bases
and 3-codon bases.
When the wobble nucleoside is inosine, the tRNA can base-
pair with three codons those ending in A, C or U.

CP 10.3: menjelaskan aspek-aspek sintesis protein

Hipotesis Wobble : Diperlukan 31 antikodon
untuk membaca 61 kodon
Sandi Genetik Hampir Universal
Keuniversalan sandi genetik terlihat dari
kesamaan sandi antara berbagai spesies,
misal antara bakteri dan tumbuhan
Ketidak universalan terlihat bahwa antara
gen mitokondria dengan gen inti terdapat
perbedaan sandi genetik
Sandi Genetik Mitokondria Khamir
Sandi Genetik Mitokondria Mamalia
 ASPECTS OF PROTEIN SYNTHESIS

Ribosome binding site

The ribosome binding site is a sequence just upstream of
the initiation codon in prokaryotic mRNA which base-pairs
with a complementary sequence near the 3-end of the 16S
rRNA to position the ribosome for initiation of protein
synthesis.
It is also known as the ShineDalgarno sequence after its
discoverers.

CP 10.3: menjelaskan aspek-aspek sintesis protein

 ASPECTS OF PROTEIN SYNTHESIS

Polysomes
Polyribosomes (polysomes) form on an mRNA when
successive ribosomes attach, begin translating and move
along the mRNA.
A polysome is a complex of multiple ribosomes in various
stages of translation on one mRNA molecule.

CP 10.3: menjelaskan aspek-aspek sintesis protein

 ASPECTS OF PROTEIN SYNTHESIS
Initiator RNA
A special tRNA (initiator tRNA), recognizing the AUG start
codon, is used to initiate protein synthesis in both
prokaryotes and eukaryotes.
In prokaryotes, the initiator tRNA is first charged with
methionine by methionyl-tRNA synthetase.
The methionine residue is then converted to N-
formylmethionine by transformylase. In eukaryotes, the
methionine on the initiator tRNA is not modified.
There are structural differences between the E. coli initiator
tRNA and the tRNA that inserts internal Met residues.

CP 10.3: menjelaskan aspek-aspek sintesis protein

 ASPECTS OF PROTEIN SYNTHESIS
Initiator RNA

CP 10.3: menjelaskan aspek-aspek sintesis protein

MECHANISM OF PROTEIN
SYNTHESIS
 MECHANISM OF PROTEIN SYNTHESIS
Overview
There are three stages of protein synthesis:
initiation the assembly of a ribosome on an mRNA;
elongation repeated cycles of amino acid delivery,
peptide bond formation and movement along the mRNA
(translocation);
termination the release of the polypeptide chain.

CP 10.4: menjelaskan meknisme sintesis protein

 MECHANISM OF PROTEIN SYNTHESIS
Initiation
In prokaryotes, initiation requires the large and small
ribosome subunits, the mRNA, the initiator tRNA, three
initiation factors (IFs) and GTP.
IF1 and IF3 bind to the 30S subunit and prevent the large
subunit binding. IF2 + GTP can then bind and will help the
initiator tRNA to bind later.
This small subunit complex can now attach to an mRNA via
its ribosome-binding site.
The initiator tRNA can then base-pair with the AUG initiation
codon which releases IF3, thus creating the 30S initiation
complex.
The large subunit then binds, displacing IF1 and IF2 + GDP,
giving the 70S initiation complex which is the fully
assembled ribosome at the correct position on the mRNA.
CP 10.4: menjelaskan meknisme sintesis protein
 MECHANISM OF PROTEIN SYNTHESIS
Initiation

CP 10.4: menjelaskan meknisme sintesis protein

 MECHANISM OF PROTEIN SYNTHESIS
Elongation
Elongation involves the three factors (EFs), EF-Tu, EF-Ts
and EF-G, GTP, charged tRNAs and the 70S initiation
complex (or its equivalent).
It takes place in three steps.
A charged tRNA is delivered as a complex with EF-Tu
and GTP. The GTP is hydrolyzed and EF-Tu:GDP is
released which can be re-used with the help of EF-Ts
and GTP (via the EF-TuEF-Ts exchange cycle).
Peptidyl transferase makes a peptide bond by joining
the two adjacent amino acids without the input of more
energy.
Translocase (EF-G), with energy from GTP, moves the
ribosome one codon along the mRNA, ejecting the
uncharged tRNA and transferring the growing peptide
chain to the P-site.
CP 10.4: menjelaskan meknisme sintesis protein
 MECHANISM OF PROTEIN SYNTHESIS
Elongation

CP 10.4: menjelaskan meknisme sintesis protein

 MECHANISM OF PROTEIN SYNTHESIS
Termination
Release factors (RF1 or RF2)
recognize the stop codons and,
helped by RF3, make peptidyl
transferase join the polypeptide
chain to a water molecule, thus
releasing it.
Ribosome release factor helps
to dissociate the ribosome
subunits from the mRNA.

CP 10.4: menjelaskan meknisme sintesis protein

Struktur dan Fungsi Situs enzim
Ribosom peptidil-
transferase

Situs mRNA :
mRNA dikenali
oleh rRNA16S
yang terdapat
pada subunit Situs A:
kecil tempat
Situs P: tempat
peptidil-tRNA aminoasil-
tRNA.
Struktur dan Fungsi
Ribosom

Situs A:
Situs P: tempat tempat
peptidil-tRNA aminoasil-
tRNA.
 Insiasi Translasi
Dimulai dengan penempelan subunit kecil ribosom kecil
pada situs Shine Dalgarno, penempelan tRNA-met
inisiator pada kodon awal (situs P), dan pempelan
subunit besar ribosom

SD

30S Kodon
awal

Kompleks
translasi
Intensitas Inisiasi :
ditentukan oleh
keserasian Shine
Dalgarno- rRNA16S
Aminoasil
-tRNA
Sintesis
Perpanjangan
Polipeptida
Amino asil-tRNA
Situs A masuk ke situs A,
Perangkaian
asam-amino dari
situs P ke situs A,
Pergeseran
ribosom
membaca kodon
berikutnya
 Reaksi Transpeptidasi

Situs P Situs A

Asam amino/peptida di situs P dilepas dari

tRNA dan disambungkan ke asam amino di
Asam
Proses Akhir Translasi
amino Polipeptida

Kodon akhir
Bila ribosom mencapai kodon akhir tidak ada
tRNA yang cocok. Akan masuk RF di situs A,
reaksi dengan H2O, dan pembebasan
polipeptida, mRNA, tRNA dan ribosom
 Next....

Gene manipulation