RNA Synthesis (Transcription)

Cont----
• It is the process of production of RNA copy
from a specific region along the length of DNA
• The structure of DNA is not altered as a result
of this process, and it continues to store
information.
• Genes are defined as DNA sequences that are
transcribed into RNA
 Cont----
• Each gene has a promoter and one or more
regulatory sequences.
– The promoter “promotes” transcription
– The regulatory sequences control when and where
(in what cell type) the gene will be expresssed
• The promoter and the regulatory sequences
are DNA sequences that are not part of the
transcript. They are recognized and bound by
DNA-binding proteins.
 Transcription unit
• The transcription unit is a sequence (stretch)
of DNA, which is formed of the gene proper,
the transcription initiation and basal rate
controlling sequence (the promoter), the
regulatory gene response sequences
(enhancer/silencer) and the transcription
termination sequence.
 Cont----
• The gene enhancer/silencer, are the gene response
sequences that control the regulated rate of gene
expression (more than the basal level).
• Transcribed region or gene proper, the DNA
sequence that is copied as hnRNA or other types of
RNAs.
• Termination region, a regulatory DNA sequence
down-stream the gene proper of some genes, at
which the RNA polymerase disassembles from the
DNA template.
 Promoters
• Are DNA sequences that “promote” gene expression
• More precisely, they direct the exact location for the
initiation of transcription
• Promoters are typically located just “upstream” (5’) of
the site where transcription of a gene actually begins
• The promoter attracts RNA polymerase, the enzyme
responsible for transcribing RNA, to the gene.
Without a promoter, a gene sequence would not be
transcribed.
 The gene is located on the coding strand

TEMPLATE

CODING
 Requirments
• 1. Template
• RNA is fundamentally single-stranded and
therefore only one strand of the DNA is actually
copied into RNA during transcription.
•  The strand that is actually being copied is termed
the template strand.
• The RNA transcript will have the opposite polarity
and the complementary sequence to this strand
•  The opposite strand is called the coding strand
 Cont----
• The base sequence of this strand is identical in
polarity and sequence to the RNA transcript
– Except for the substitution of uracil in RNA
for thymine in DNA
• Because the coding strand has the same
sequence and polarity as the RNA, it is said to
“carry the gene.”
 Cont----
• 2. Substrates
• The substrates for RNA synthesis are the four
ribonucleotide phosphates
• ATP
• CTP
• GTP
• UTP
 Cont----
• 3. Enzyme
• RNA polymerase(DNA dependent RNA
polymerase)
• In Prokaryotes
• A single type that is responsible for synthesis
of all types of RNA
• Products of RNA polymerase require slight or
no modification after transcription.
 Cont----
• The holoenzyme is formed of five subunits:
two identical  subunits, two similar but not
identical ' subunits and a regulatory 
subunit (2').
• The core part of the enzyme is formed of the
four 2' subunits.
 Cont----
• In Eukaryotes
• There are three types; each is specific for
synthesis of a specific type of RNA.
• Mostly require extensive post-transcriptional
modifications particularly mRNA.
• They are much complex in structure and are
formed of up to 16 subunits.
 Cont----
• The three types of RNA polymerases are:
• RNA polymerase I: Responsible for synthesis of
the large RNA molecules (rRNA).
• RNA polymerase II: Responsible for synthesis
of mRNA
• RNA polymerase III: Responsible for synthesis
of small RNA molecules, i.e., tRNA and 5S
rRNA
 Stages of replication
• I- Initiation:
• Initiation occur on a single strand of a
transcription unit (a gene) that is called
template (non-coding) strand that is
complementary to the RNA, and it never occurs
in the other strand, coding strand. Coding
strand is similar to mRNA sequence except for
U/T. The template strand is read in 3'5' so that
the synthesized RNA will be formed in 5'3'.
 Cont----
• Which DNA strand is the template and which is the
coding, differs from one gene to the other but is
always the strand that contain the prompter
sequence read in 3'5' direction.
• RNA polymerase recognizes the promoter region by
the help of the  (sigma) factor.
• Then, the core enzyme binds tightly to the DNA.
Once the core molecule binds to the DNA, it
unwinds 17 nucleotides to separate the two strands.
 Cont----
• The binding of the enzyme to DNA occurs in a
sequential process, i.e.,  factor then core enzyme
(2') binds and it searches for the transcription
initiation site (an open reading frame starting at TAC).
• The synthesized RNA always starts with a purine that
enters at the initiation site of the enzyme. This purine
ribonucleotide stays in prokaryotic mature mRNA;
whereas, it is removed in mature eukaryotic mRNA
due to the post-transcriptional processing before
capping.
 Cont----
• The tight binding of the RNA polymerase to the
promoter forms what is called the closed
complex
• Then, the open complex is formed when RNA
polymerase denatures the double-stranded
DNA in the AT-rich Pribnow Box
• Next, the RNA polymerase makes a short RNA
strand copy of the template strand within the
denatured region
 Cont----
– The sigma factor is released at this point
– This marks the end of initiation
– Note that RNA polymerase, unlike DNA
polymerase, is a “smart enzyme”! It can start an
RNA strand all on its own.
• The core enzyme now slides down the DNA to
synthesize the transcript
 2. Elongation

•  The RNA transcript is synthesized during the

elongation step
• The open complex formed by the action of RNA
polymerase is about 17 bases long and remains
that size as the polymerase moves along the DNA
• Behind the open complex, the DNA rewinds back
into the double helix
• On average, the rate of RNA synthesis is about 43
nucleotides per second
 Cont----
•  Factor is released before elongation starts.
• The four ribonucleotides triphosphate; ATP,
GTP, CTP and UTP continue to enter into the
polymerization (or elongation) site of  unit
with the release of a pyrophosphate (PPi) each
time a new nucleotide is added to the growing
RNA chain.
 Cont----
• The RNA polymerase continues transcription
from 3' towards 5' end of the template strand
according to the base pairing role in an anti-
parallel manner so that A-U, G-C, T-A and C-G
• Elongation continues till the termination
point is reached.
 Cont----
• 3. Termination of transcription:
• Termination may be rho () factor-dependent or
independent.
• rho-dependent termination,
• rho factor recognizes and binds the termination
sequence in the template DNA, then; it
disassembles the enzyme/RNA/DNA complex to
release RNA polymerase and the synthesized RNA
molecule from DNA template strand
 Cont----
• rho-independent termination
• RNA polymerase stops transcription when the
synthesized complete RNA molecule takes its
three-dimensional form with the formation of
specific secondary structures such as a hairpin
• This leads to pausing of RNA polymerase and
hence disassembly of the transcription
machinery.
 Antibiotic inhibitors of transcription:

• Rifamycin: Binds to the core enzyme

occupying substrate binding site and inhibiting
the incoming nucleotides from binding to the
initiation site.
• Actinomycin D: Binds DNA template inhibiting
its transcription by preventing movement of
RNA polymerase along DNA.
 Post-transcriptional modification of RNA:

• I. Processing of mRNA:
• Eukaryotic crude transcript of mRNA produced
in the nucleus is called heterogeneous nuclear
RNA (hnRNA), i.e., the blue script.
• Post-transcriptional Processing of mRNA
include decreasing its size, 5'-capping and 3'-
tailing along with the post-maturation mRNA
editing.
 Cont----
• Intron removal (Decrease in size):
• It is due to the splicing or removal of internal
non-translatable sequences, i.e., Introns from
the translatable sequences, i.e., Exons, by
splicosome
• Removal of introns facilitates the transport of
mature mRNA from the nucleus to the
cytoplasm otherwise it will be degraded in the
nucleus.
 Cont----

Exon1 Intron1 Exon2 Intron2 Exon3

Intron1 Intron2

Exon1 Exon2 Exon3

 Cont----
• Addition of 7-methyl-guanylate 5'-capping:
• It is the addition of 7-methyl guanosine
triphosphate to the 5'-end of mRNA by
specific enzyme called guanylatetransferase.
• The cap is attached by 5' to 5' triphosphate
linkage. It enhances the translation of mRNA
and protects mRNA from the action of 5'3'
exonucleases and phosphatases.
 Cont----
CH3 O
7-methyl-gaunine
N NH
NH2
O 5' O
N N

H H
H H adenine
NH2
OH OH N N
O O
O P O P O P O
5' N N
O
O- O- O-
cytosine
H H NH2
H H
N
O O-CH3
O
O P O N
O
O- H H
H H
O O-CH3
3'O P OH
O-
 Cont----
• Addition of polyadenylate 3'-tailing:
• Addition of 20 - 250 polyadenylate tail at the
3'-end by the action of poly-A-polymerase
enzyme. It is specified by a special sequence
near the 3'-end of the mRNA (AAUAAA).
• The tail protects 3'-end of mRNA from 3'5'
exonuclease and facilitates mRNA transport
into cytoplasm.
 II- Processing of tRNA:
• Primary tRNA transcript is a large precursor
containing more than one tRNA. It is
processed by the action of specific class of
ribonucleases that also include removal of
introns and cleavage of a 5'-leader sequence.
• The tRNA nucleotides are modified by
methylation, deamination, alkylation,
reduction and glycosylation.
 Cont----
• Finally, replacement of the 3'-terminal UU by
the characteristic amino acceptor CCA
terminus at the 3' ends by
nucleotidyltransferase.
• This end of tRNA function as an acceptor arm
for binding amino acids.
 III- Processing of ribosomal RNA:
• A large 45S precursor intron-less molecule is
cleaved by specific endonuclease and
exonuclease into 5.8S, 18S, and 28S rRNAs.
The 5S rRNA is a separate gene.
• The 5S, 5.8S and 28S together with 45 proteins
form the 60S sub unit and the 18S together
with 33 proteins form the 40s sub unit of the
rRNA.
 Genetic code
• It is the collection of codons
• Codon is a three base sequence in DNA that is copied
in mRNA (with U/T) which determines the type and
site of amino acid in the translated protein.
• It is usually read in the 5'3' direction in the mRNA.
• It is identified according to base complementarity in
an anti-parallel manner by the anticodon, present in
tRNA (3'5' direction).
 Cont-----
• All probable triple alternative combinations of
the four bases enter in DNA or RNA structure give
64 triple codons or possible amino acid names.
• Among the 64 codons there are three codons
(UAA, UAG, UGA) that are called non-sense (i.e.,
meaning-less) or Termination codons that is
because they do not code for an amino acid but
they signify termination of translation of mRNA.
 Cont-----
Characteristics of the genetic code:
• Specific or unambiguous since each codon is
specific for a single amino acid, e.g., UUU
encodes phenylalanine only.
• Degenerate, since there are 61 amino acid
encoding codons, each amino acid may be
encoded for by several codons. The 61 codons
are not equally divided on the 20 amino acids
entering in protein structure.
 Cont----
• Therefore, arginine has 6 codons (synonyms or
nicknames, CGU, CGC, CGA, CGG, AGA and
AGG), whereas, methionine has only one
(AUG). This is why codon is said to be
degenerate, see the above table.
• Universality: The genetic code is universal,
i.e., specify the same amino acid in all living
organisms from viruses, bacteria, plants,
insects to mammals.
 Cont------
• Non-overlapping: The mRNA codons are read
in a continuous manner in the 5'3' direction
without interruption in three-base sequence,
i.e., no base functions as a common member
of two consecutive codons. Therefore,
addition or removal of a base leads to shifting
of the reading frame producing totally
different amino acid sequence.