PROTEIN SYNTHESIS(Transcription)
Musonda Machiko
Dip. Biomed( EHC), Bsc. Biomed(UNZA)
[email protected]
2
Introduction
Protein synthesis is the process in which
cells build proteins. The term is
sometimes used to refer only to protein
translation but more often it refers to a
multi-step process, beginning with
amino acid synthesis and transcription
of nuclear DNA into messenger RNA,
which is then used as input to
translation.
STEPS OF PROTEIN SYNTHESIS
1. TRANSCRIPTION
2. TRANSLATION
3. POST-TRANSLATION
TRANSCRIPTION
Transcription is the first step of gene expression, in which a particular
segment of DNA is copied into RNA by the enzyme, RNA polymerase.
The stretch of DNA transcribed into an RNA molecule is called
a transcription unit and encodes at least one gene. If the gene
transcribed encodes a protein, the result of transcription is
messenger RNA (mRNA), which will then be used to create that
protein via the process of translation. Alternatively, the transcribed
gene may encode for either non-coding RNA genes (such as microRNA
, lincRNA, etc.) or ribosomal RNA (rRNA) or transfer RNA (tRNA), other
components of the protein-assembly process, or other ribozymes.
A DNA transcription unit encoding for a
protein contains not only the sequence that
will eventually be directly translated into the
protein (the coding sequence) but
also regulatory sequences that direct and
regulate the synthesis of that protein. The
regulatory sequence before the coding
sequence is called the five prime
untranslated region (5'UTR), and the
sequence following the coding sequence is
called the three prime untranslated region
(3'UTR).
As in DNA replication, DNA is read from 3'UTR → 5'UTR
during transcription. Meanwhile, the complementary
RNA is created from the 5'UTR → 3'UTR direction. This
means its 5' end is created first in base pairing. Although
DNA is arranged as two antiparallel strands in a
double helix, only one of the two DNA strands, called the
template strand, is used for transcription. This is because
RNA is only single-stranded, as opposed to double-
stranded DNA. The other DNA strand is called the coding
(lagging) strand, because its sequence is the same as the
newly created RNA transcript (except for the substitution
of uracil for thymine). The use of only the 3'UTR → 5'UTR
strand eliminates the need for the Okazaki fragments
seen in DNA replication.
PROKARYOTIC RNA POLYMERASE
(one type)
There are two main segments of the RNA polymerase molecule:
I. The core enzyme(1 beta,1 beta prime & 2 alpha sub units)
II. sigma subunit
Core enzyme + sigma subunit=HOLOENZYME
The sigma subunit of RNA polymerase is the part of the enzyme
responsible for recognizing the signal on the DNA strand that tells
the polymerase to begin synthesizing RNA. It is through this sigma
unit that RNA polymerase is able to initiate transcription.
EUKARYOTIC RNA POLYMERASE
(3 TYPES)
1. RNA polymerase I makes rRNA
2. RNA polymerase II makes mRNA
3. RNA polymerase III makes tRNA
Major steps of Transcription
1. Initiation
Core Promoters are regions of DNA that promote transcription and RNA polymerase is able to bind to
core promoters in the presence of various specific transcription factors. The most characterized type of
core promoter in eukaryotes is a short DNA sequence known as a TATA box which is a binding site for a
transcription factor known as TATA-binding protein (TBP), which is itself a subunit of another
transcription factor, called Transcription Factor II D (TFIID).
Transcription factor II H, has two components with helicase activity and so is involved in the separating
of opposing strands of double-stranded DNA to form the initial transcription bubble.
In Prokaryotes, transcription begins
with the binding of RNA polymerase
to the promoter in DNA with the
help of the sigma factor subunit.
2. Elongation
One strand of the DNA, the template strand( non
coding), is used as a template for RNA synthesis. As
transcription proceeds, RNA polymerase uses base
pairing complementarity with the DNA template to
create an RNA copy.
This produces an RNA molecule from 5' → 3', an
exact copy of the coding strand (except that
thymines are replaced with uracils, and the
nucleotides are composed of a ribose (5-carbon)
sugar where DNA has deoxyribose
3. Termination
Prokaryotes use two different strategies for transcription
termination:
I. Rho-dependent termination
A protein called Rho comes to disturbilize the interraction
between Rna polymerase and template strand thereby
signaling transcription termination.
ii. Rho-independent transcription termination
also called intrinsic termination, RNA transcription stops
when the newly synthesized RNA molecule forms a G-C-
rich hairpin loop followed by a run of Us. When the
hairpin forms, the mechanical stress breaks the bonds
Transcription termination in
eukaryotes is less understood but
involves cleavage of the new transcript
followed by template-independent
addition of As at its new 3' end, in a
process called polyadenylation.
Messenger RNA processing
In prokaryotes, no RNA processing is necessary:
– the nascent RNA is usually the mRNA.
• In eukaryotes, the nascent RNA is called primary
transcript-RNA
– needs to be processed
– and transported to the cytoplasm for translation to
occur.
The processing steps are:
– Addition of a 5’ 7-methyl guanosine
cap (capping).
– Addition of a poly-A tail at the 3’ end
(polyadenylation)
– RNA splicing to remove intervening
sequences (remove
introns).
Eukaryotic RNA Processing: Capping
When the RNA chain is about 30 nucleotides
long, the 5’ ends are
modified by the addition of a guanine group
Methyl transferases then add methyl groups in
the 7 position to
that and a couple more nucleotides.
• The caps are recognized by the translation
machinery.
• They protect the growing RNA chain from
degradation by nucleases
Eukaryotic RNA Processing:
Polyadenylation
nascent RNA is cleaved downstream from the AAUAAA
conserved sequence.
– By ribonucleaseThe enzyme poly(A) polymerase adds
adenine
ribonucleotides
– up to 200 bases long at the 3’ end of the RNA.
• The poly(A) tail
– enhances the stability of eukaryotic mRNA and
– regulates its transport to the cytoplasmic compartment.
Eukaryotic RNA Processing:
RNA splicing
A “Simple” Eukaryotic Gene
Transcription
5’ UTR 3’ UTR
Start Site
Introns
5’ Exon 1 Int. 1 Exon 2 Int. 2 Exon 3 3’
Promoter/ Exons Terminator
Control Region Sequence
Transcription
5’ Exon 1 Int. 1 Exon 2 Int. 2 Exon 3 3’
Unprocessed RNA Transcript
Processing of eukaryotic mRNA
5’ UTR 3’ UTR
Protein Coding Region
G Exon 1 Exon 2 Exon 3 AAAAA
5’ Cap 3’ Poly A Tail
t.1 t.2
In In
• RNA processing achieves three things:
1) Removal of Introns
2) Addition of a 5’ cap
3) Addition of a 3’ tail
The mRNA then moves out of the nucleus and is
translated in the cytoplasm.
Ready for Translation………
