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OVERVIEW OF
TRANSCRIPTION
V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, India.
[email protected] Also available at https://tvuni.academia.edu/mvinayagam
TranscriptionTranscription is the first step of gene
expression, in which a particular segment of
DNA is copied into RNA by the enzyme RNA
polymerase. Both RNA and DNA are nucleic
acids, which use base pairs of nucleotides as a
complementary language. The two can be
converted back and forth from DNA to RNA by
the action of the correct enzymes. During
transcription, a DNA sequence is read by an
RNA polymerase, which produces a
complementary, antiparallel RNA strand called
a primary transcript.
Transcription proceeds in the following
general steps:
1. One or more sigma factor protein binds to
the RNA polymerase holoenzyme, allowing it to
bind to promoter DNA.
2. RNA polymerase creates a transcription
bubble, which separates the two strands of the
DNA helix. This is done by breaking the
hydrogen bonds between complementary DNA
nucleotides.
3. RNA polymerase adds matching RNA
nucleotides to the complementary nucleotides
of one DNA strand.
4. RNA sugar-phosphate backbone forms with
assistance from RNA polymerase to form an
RNA strand.
5. Hydrogen bonds of the untwisted RNA-DNA
helix break, freeing the newly synthesized RNA
strand.
6. If the cell has a nucleus, the RNA may be further
processed. This may include polyadenylation,
capping, and splicing.
7. The RNA may remain in the nucleus or exit to
the cytoplasm through the nuclear pore complex.
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, messenger RNA (mRNA) will be
transcribed; the mRNA will in turn serve as a
template for the protein synthesis through
translation. Alternatively, the transcribed gene
may encode for either non-coding RNA (such as
micro RNA), ribosomal RNA (rRNA), transfer RNA
(tRNA), or other ribozymes. Overall, RNA helps
synthesize, regulate, and process proteins; it
therefore plays fundamental role in performing
functions within a cell.
Regulation of transcription
A gene consists of a transcriptional region and a
regulatory region. The transcriptional region is the
part of DNA to be transcribed into a primary
transcript (an RNA molecule complementary to the
transcriptional region). The regulatory region can
be divided into cis-regulatory (or cis-acting)
elements and trans-regulatory (or trans-acting)
elements. The cis-regulatory elements are the
binding sites of transcription factors which are the
proteins that, upon binding with cis-regulatory
elements, can affect (either enhance or repress)
transcription. The trans-regulatory elements are
the DNA sequences that encode transcription
factors.
Transcription factor
In molecular biology and genetics, a
transcription factor (sequence-specific DNA-
binding factor) is a protein that binds to
specific DNA sequences, thereby controlling
the rate of transcription of genetic information
from DNA to messenger RNA. Transcription
factors perform this function alone or with
other proteins in a complex, by promoting (as
an activator), or blocking (as a repressor) the
recruitment of RNA polymerase (the enzyme
that performs the transcription of genetic
information from DNA to RNA) to specific
genes.
ActivatorsActivators enhance the interaction between
RNA polymerase and a particular promoter,
encouraging the expression of the gene.
Activators do this by increasing the attraction
of RNA polymerase for the promoter, through
interactions with subunits of the RNA
polymerase or indirectly by changing the
structure of the DNA.
RepressorsRepressors bind to non-coding sequences on
the DNA strand that are close to or overlapping
the promoter region, inhibiting RNA
polymerase's progress along the strand, thus
blocking the expression of the gene.
EnhancerIn genetics, an enhancer is a short (50-1500
bp) region of DNA that can be bound with
proteins (activators) to activate transcription
of a gene or genes. These proteins are usually
referred to as transcription factors. Enhancers
are generally cis-acting, located up to 1 Mbp
away from the gene. There are hundreds of
thousands of enhancers in the human genome.
Enhancers are sites on the DNA helix that are
bound to by activators in order to loop the
DNA bringing a specific promoter to the
initiation complex.
Silencers
Silencers are regions of DNA that are bound by
transcription factors in order to silence gene
expression. The mechanism is very similar to that
of enhancers. In genetics, a silencer is a DNA
sequence capable of binding transcription
regulation factors, called repressors. DNA
contains genes and provides the template to
produce messenger RNA (mRNA). That mRNA is
then translated into proteins that activate or
inactivate gene expression in cells. When a
repressor protein binds to the silencer region of
DNA, RNA polymerase—the enzyme that
transcribes DNA into RNA—is prevented from
binding to the promoter region. With the
transcription of DNA into RNA blocked, the
translation of RNA into proteins is impossible.
Thus, silencers prevent genes from being
expressed as proteins.
Specificity factors alter the specificity of RNA
polymerase for a given promoter or set of
promoters, making it more or less likely to bind
to them (i.e. sigma factors used in prokaryotic
transcription).
A sigma factor (σ factor) is a protein needed only
for initiation of RNA synthesis. It is a bacterial
transcription initiation factor that enables
specific binding of RNA polymerase to gene
promoters. The specific sigma factor used to
initiate transcription of a given gene will vary,
depending on the gene and on the environmental
signals needed to initiate transcription of that
gene.
PromoterIn genetics, a promoter is a region of DNA that
initiates transcription of a particular gene.
Promoters are located near the transcription start
sites of genes, on the same strand and upstream
on the DNA (towards the 5' region of the sense
strand). Promoters can be about 100–1000 base
pairs long.
A core enzyme consists of the subunits of an
enzyme that are needed for catalytic activity, as in
the core enzyme RNA polymerase. RNA
polymerase is a core enzyme consisting of five
subunits: 2 α subunits, 1 β subunit, 1 β‘ subunit,
and 1 ω subunit. At the start of initiation, the core
enzyme is associated with a sigma factor that aids
in finding the appropriate -35 and -10 base pairs
downstream of promoter sequences. When the
sigma factor and RNA polymerase combine, they
form a holoenzyme.
Inducers
In molecular biology, an inducer is a molecule
that starts gene expression. An inducer can
bind to repressors or activators.
Inducers function by disabling repressors. The
gene is expressed because an inducer binds to
the repressor. The binding of the inducer to
the repressor prevents the repressor from
binding to the operator. RNA polymerase can
then begin to transcribe operon genes.
Transcriptional repressors
Transcriptional repressors are proteins that bind to
specific sites on DNA and prevent transcription of
nearby genes. (RNA can also inhibit transcription,
but inhibitory RNAs are not usually called
repressors). In molecular genetics, a repressor is a
DNA- or RNA-binding protein
that inhibits the expression of one or more genes by
binding to the operator. A DNA-binding repressor
blocks the attachment of RNA polymerase to the
promoter, thus preventing transcription of the genes
into messenger RNA. An RNA-binding repressor
binds to the mRNA and prevents translation of the
mRNA into protein. This blocking of expression is
called repression. A defining feature of transcription
factors is that they contain one or more DNA binding
domains (DBDs), which attach to specific sequences
of DNA adjacent to the genes that they regulate.
transcription – the process of making RNA from
a DNA template by RNA polymerase
factor – a substance, such as a protein, that
contributes to the cause of a specific
biochemical reaction or bodily process
transcriptional regulation – controlling the rate
of gene transcription for example by helping or
hindering RNA polymerase binding to DNA
upregulation, activation, or promotion – increase
the rate of gene transcription
downregulation, repression, or suppression –
decrease the rate of gene transcription
coactivator – a protein that works with
transcription factors to increase the rate of gene
transcription
corepressor – a protein that works with
transcription factors to decrease the rate of
gene transcription
PROKARYOTIC
TRANSCRIPTION
PROKARYOTIC TRANSCRIPTION
Transcription is the first step of
gene expression, in which a particular
segment of DNA is copied into RNA (
mRNA) by the enzyme RNA polymerase.
Simply stated transcription is the
synthesis of RNA from a DNA template or
The flow of genetic information from DNA
to RNA or synthesis single stranded RNA
from double stranded DNA. All the three
RNAs- tRNA, mRNA, rRNA are
synthesized form the DNA by DNA
dependent RNA polymerase.
RNA POLYMERASE
The E.Coli RNA polymerase is one of the largest
enzyme in the cell. The enzyme consist of five
subunits. These are alpha α, beta β, beta prime β',
omega and sigma.
Two alpha subunits:
Essential for assembly of the enzyme activation by
some regulatory proteins
These two identical alpha subunit play role in
promotor recognition
Βeta subunit
It is the catalytic centre of RNA polymerase and has
two domains responsible for transcription initation
and elongation. Beta subunit binds the nucleotide
triphosphate (NTP) subtrates and interacts with
sigma.
Βeta prime subunit
Larghest subunit functions in DNA binding, this
subunit binds two Zn2+ ions which are thought
to participate in the catalytic function of the
polymerase.
Sigma subunit
The most common sigma factor in E.Coli of
sigma -70 (molecular mass 70kDa). Binding of
sigma factor converts the core enzyme into
RNA polymerase holo enzyme. Sigma factor
critical role in promotor recognition, but it is
not required for elongation.
The sigma factor contributes to promotor
recognition by decreasing the affinity of the core
enzymes for non specific DNA sites and increasing
the affinity for the promotor.
Like DNA polymerase RNA polymerase links
ribonucleotide 5’ triphosphates (ATP,GTP,CTP,UTP)
in an order specified by base pairing with a
template. The ribonucleotides are linked through 3’
– 5’ phosphor diester bond formed by the attach of
5’ alpha phosphate of one ribonucleotide to the 3’
OH group of adjacent ribonucleotide.
The enzyme RNA polymerase moves along a DNA
template strand in the 3’-5’ direction joining the 5’
phosphate of an incoming ribonucleotide to the 3’-
OH of the previous residue. Thus the RNA chain
grows 5’- 3’ during transcription. The reaction is
driven by subsequent hydrolysis of PPi to inorganic
phosphate by ubiquitoes pyrophosphate activity.
Three steps in transcription
Initiation
Elongation
Termination
Initiation
Initiation begins with the sigma subunit of
RNA polymerase recognizes the promotor
sequence, and binding of DNA dependent RNA
polymerase holoenzyme to promoter in template
of DNA forms closed promotor complex.
In genetics, a promoter is a region of DNA that
initiates transcription of a particular gene.
Promoters are located near the transcription
start sites of genes, on the same strand and
upstream on the DNA (towards the 5' region of
the sense strand). Promoters can be about 100–
1000 base pairs long
Once the closed promotor complex is
established, the RNA ploymerase holo enzyme
unwinds about 14 base pairs of DNA (base pair
located at –10 to + 2 relative to the
transcription start site) forming a very stable
open promotor complex. In this comples RNA
polymerase holo enzyme bound very tightly to
the DNA.
The -35 region and the -10 ("Pribnow box")
region comprise the core prokaryotic promoter,
and |T| stands for the terminator. The DNA on
the template strand between the +1 site and the
terminator is transcribed into RNA, which is
then translated into protein. At this stage, the
DNA is double-stranded ("closed"). This
holoenzyme/wound-DNA structure is referred to
as the closed complex.
-10 sequence/Pribnow box/TATA box/ Hogness
box – it contain six nucleotide (TATAAT) located
8 to 10 nucleotide to the left of transcriptional
start site. The – 10 region important for DNA
unwinding.
35 region - it contain six nucleotide
(TTGACA), this sequence is separated from -10
box by 19 bp.
In order to transcription to begin, the DNA
duplex must be “opened” so that RNA
polymerase has assess to single stranded
template.
The RNAP sigma subunit is directly
involved in melting the DS-DNA .
Interaction of the sigma subunit with the
non template strand maintains the open
complex.
Human as 105 initiation sites. RNAP first
scans DNA at 10-3 bp/s until it finds
(specially sigma factor) promoter
sequences to which it binds firmly.
Promoters are present in coding strand in
5’ to 3’ direction.
Elongation
Once the promoters region has been recognized
by sigma factor of holoenzyme the enzyme
begins to synthesis RNA sequence, sigma factor
is released. This enzyme has no exo/endo
nuclease activity and cannot repair the
mistakes as DNA polymerase in replication.
RNA polymerase add complementary base to
the template strand of DNA. It adds Thiamine
for Adenine (T =A), Guanine for Cytosine (G ≡
C), Cytosine for Guanine (C ≡ G) and Adenine
for Uracil (A = U).
Most transcripts originate using adenosine-5'-
triphosphate (ATP) and, to a lesser extent,
guanosine-5'-triphosphate (GTP) (purine
nucleoside triphosphates) at the +1 site.
Uridine-5'-triphosphate (UTP) and cytidine-5'-
triphosphate (CTP) (pyrimidine nucleoside
triphosphates) are disfavoured at the initiation
site.
The dissociation of σ allows the core RNA
polymerase enzyme to proceed along the DNA
template, synthesizing mRNA in the 5' to 3'
direction at a rate of approximately 40 nucleotides
per second. As elongation proceeds, the DNA is
continuously unwound ahead of the core enzyme
and rewound behind it . Since the base pairing
between DNA and RNA is not stable enough to
maintain the stability of the mRNA synthesis
components, RNA polymerase acts as a stable
linker between the DNA template and the nascent
RNA strands to ensure that elongation is not
interrupted prematurely.
TERMINATION
E. coli has 2 class of termination sequence in
template DNA. One class is recognized by
termination protein "Rho" ,that's rho-
dependent and other is rho independent.
a. Rho-independent.
Formation of RNA transcript with pallindromic
sequence (self complementary) that form
hairpin structure (GC rich) and another
structure is conserved string of 3A residue in 3’
end of template strand.
b. Rho-dependent:
Rho protein associates with RNA at C-rich
site near 3’ end and moves along the RNA
until it reaches RNAP paused at termination
site. The rho protein has ATP dependent
RNA-DNA helicase activity that promotes
release of RNA-DNA hybrid helix causing the
release of RNA. In eukaryotic cell after 3’
end of transcript is encoded, RNA
endonuclease cleaves the primary transcript
about 15 bases 3’ to consensus sequence
AAUAAA that serves as cleavage signal.
Action of antibiotics:
Rifampin (anti tuberculosis drug) -
inhibits the initiation of transcription by
binding to the β subunit of prokaryotic
RNA polymerase, thus interfering with the
formation of the first phosphodiester
bond.
Dactinomycin (Actinomycin D) –
Anti cancer drug - It binds to the DNA
template and interferes with the
movement of RNA polymerase along the
DNA
Inhibitors
EUKARYOTIC
TRANSCRIPTION
EUKARYOTIC TRANSCRIPTION
Eukaryotic transcription is the elaborate process that
eukaryotic cells use to copy genetic information stored in
DNA into units of RNA replica. A eukaryotic cell has a
nucleus that separates the processes of transcription and
translation. Eukaryotic transcription occurs within the
nucleus, where DNA is packaged into nucleosomes and
higher order chromatin structures. The complexity of the
eukaryotic genome requires a great variety and complexity
of gene expression control.
Eukaryotic transcription proceeds in three sequential
stages: initiation, elongation, and termination. The
transcriptional machinery that catalyzes this complex
reaction has at its core three multi-subunit RNA
polymerases.
Eukaryotes have three nuclear RNA polymerases, each
with distinct roles and propertiesName Location Product
RNA Polymerase I (Pol I, Pol A) nucleolus larger ribosomal RNA (rRNA) (28S, 18S, 5.8S)
RNA Polymerase II (Pol II, Pol B) Nucleus
Messenger RNA (mRNA), most small nuclear RNAs (snRNAs), small interfering RNA (siRNAs) and micro RNA (miRNA).
RNA Polymerase III (Pol III, Pol C)
nucleus (and possibly the nucleolus-nucleoplasm interface)
transfer RNA (tRNA), other small RNAs (including the small 5S ribosomal RNA (5s rRNA), snRNA U6, signal recognition particle RNA (SRP RNA) and other stable short RNAs
RNA polymerase I
RNA polymerase I (Pol I) catalyzes the transcription of all
rRNA genes except 5S rRNA.
These rRNA genes are organized into a single
transcriptional unit and are transcribed into a continuous
transcript.
This precursor is then processed into three rRNAs: 18S,
5.8S, and 28S. The transcription of rRNA genes takes place in
a specialized structure of the nucleus called the nucleolus,
where the transcribed rRNAs are combined with proteins to
form ribosomes.
Promoter Structure: For RNA pol-I:
Genes for ribosomal RNA are exclusively transcribed by
RNA polymerase-I.
In eukaryotic system most active and highly productive
genes, which are transcribed most of the time, are ribosomal
RNA genes.
More than 90 % of the total RNA found in any eukaryotic
cell is rRNA.
Its synthesis is triggered, when cells are activated for cell
proliferation, in such situations tremendous increase of
rRNA takes place, ex. rRNA synthesis during oogenesis is a
par excellent example.
Initiation
It has, what is termed as core promoter region between (-) 10
and (-) 45 and an upstream control elements (UCE), it is the
region to which upstream element binding factors bind.
The core region attracts selectivity factor SL-I, 3 TAFs (TBP
associated factors) and TBP (TATA binding factors). Positioning
of the TBP is assisted and determined by the SL-I and then TAFs
bring TBP.
It is now known that two histone like proteins are also
associated with this complex.
This assembly ultimately brings RNA pol-I to the site. But
the activation depends on upstream control element binding
factors UBF 1; they bind not only to the core but also to UCE.
UBFI binding results in protein-protein interaction in such a
way two units of UBFs join with one another with a DNA loop,
and activate the RNA pol-I complex.
Elongation
As Pol I escapes and clears the promoter, UBF and SL1
remain-promoter bound, ready to recruit another Pol I. Indeed,
each active rDNA gene can be transcribed multiple times
simultaneously. Pol I does seem to transcribe through
nucleosomes, either bypassing or disrupting them, perhaps
assisted by chromatin-remodeling activities. In addition, UBF
might also act as positive feedback, enhancing Pol I elongation
through an anti-repressor function. An additional factor, TIF-
IC, can also stimulate the overall rate of transcription and
suppress pausing of Pol I. As Pol I proceeds along the rDNA,
supercoils form both ahead and behind the complex. These are
unwound by topoisomerase I or II at regular interval, similar to
what is seen in Pol II-mediated transcription. Elongation is
likely to be interrupted at sites of DNA damage. Transcription-
coupled repair occurs similarly to Pol II-transcribed genes and
require the presence of several DNA repair proteins, such as
TFIIH, CSB, and XPG.
Termination
In higher eukaryotes, TTF-I binds and bends the
termination site at the 3' end of the transcribed region. This
will force Pol I to pause. TTF-I, with the help of transcript-
release factor PTRF and a T-rich region, will induce Pol I
into terminating transcription and dissociating from the
DNA and the new transcript. Evidence suggests that
termination might be rate-limiting in cases of high rRNA
production. TTF-I and PTRF will then indirectly stimulate
the reinitiation of transcription by Pol I at the same rDNA
gene. In organisms such as budding yeast the process seems
to be much more complicated.
rRNA Synthesis and Processing
The genes coding for rRNA (except 5S rRNA) are located in
the nucleolar part of the nucleus. The rRNA genes are highly
repetitious and mammalian cells contain 100 to 2000 copies of
the rRNA genes per cell. The genes are organised in
transcription units separated by non-transcribed spacers. Each
transcription unit contains sequences coding for 18S, 5.8S and
28S rRNA.
The transcription units are transcribed by RNA polymerase I
into giant RNA molecules, primary transcripts, that in addition
to the sequences corresponding to 18S, 5.8S and 28S rRNA
contains external and internal transcribed spacer sequences.
The rate of nucleolar transcription is very high and many
polymerases operate on the same transcription unit. The
transciptionally active DNA therefore has a Christmas tree-
like appearance on electron microscopic pictures.
The primary transcript is processed into the mature 18S, 5.8S
and 28S rRNAs. The processing involves exo- and endo-
nucleolytic cleavages guided by snoRNA (small nucleolar
RNAs) in complex with proteins. The mature rRNAs contain
modified nucleotides which are added after transcription by a
snoRNA-dependent mechanism.
5S ribosomal RNA is transcribed by RNA polymerase III in the
nucleoplasm. Each eukaryotic cell contains a high number of
copies of the 5S coding gene (up to 20 000 copies per cell). 5S
rRNA contains overlapping binding sites for two different
proteins, ribosomal protein L5 and transcription factor TFIIIA.
The mutual exclusive binding of these two proteins to 5S rRNA
is important for coordinating the expression of 5S rRNA to the
production of the other rRNAs.
RNA polymerase II
RNA polymerase II
RNA polymerase II (RNAP II and Pol II) is an
enzyme found in eukaryotic cells. It catalyzes the transcription
of DNA to synthesize precursors of mRNA and most snRNAs,
siRNAs, and all miRNAs and microRNA. A 550 kDa complex
of 12 subunits, RNAP II is the most studied type of RNA
polymerase. A wide range of transcription factors are required
for it to bind to upstream gene promoters and begin
transcription.
Many Pol II transcripts exist transiently as single
strand precursor RNAs (pre-RNAs) that are further processed
to generate mature RNAs. For example, precursor mRNAs
(pre-mRNAs) are extensively processed before exiting into the
cytoplasm through the nuclear pore for protein translation.
Promoter RNA polymerase – II
Most eukaryotes use TATA box (it's a little further away
from initiation start area). In eukaryotes, the promoters
are a little more complex, these elements functionally
analogous to the -10 and -35 in prokaryotes, they orient
polymerase and bind proteins.
Initiation
To begin transcription, eucaryotic RNA polymerase II requires the
general transcription factors. These transcription factors are called
TFIIA, TFIIB, and so on. (A) The promoter contains a DNA
sequence called the TATA box, which is located 25 nucleotides
away from the site where transcription is initiated. (B) The TATA
box is recognized and bound by transcription factor TFIID, which
then enables the adjacent binding of TFIIB. (C) For simplicity the
DNA distortion produced by the binding of TFIID is not shown.
(D) The rest of the general transcription factors as well as the RNA
polymerase itself assemble at the promoter. (E) TFIIH uses ATP to
pry apart the double helix at the transcription start point, allowing
transcription to begin. TFIIH also phosphorylates RNA polymerase
II, releasing it from the general factors so it can begin the
elongation phase of transcription. As shown, the site of
phosphorylation is a long polypeptide tail that extends from the
polymerase molecule.
Processing of mRNA
All the primary transcripts produced in the nucleus must
undergo processing steps to produce functional RNA
molecules for export to the cytosol. We shall confine
ourselves to a view of the steps as they occur in the
processing of pre-mRNA to mRNA.
The steps:
• Synthesis of the cap. This is a stretch of three
modified nucleotides attached to the 5' end of the pre-
mRNA.
• Synthesis of the poly (A) tail. This is a stretch of
adenine nucleotides attached to the 3' end of the pre-
mRNA.
• Step-by-step removal of introns present in the
pre-mRNA and splicing of the remaining exons. This step is
required because most eukaryotic genes are split.
5' cap addition
• A 5' cap (also termed an RNA cap, an RNA 7-
methylguanosine cap, or an RNA m7G cap) is a modified guanine
nucleotide that has been added to the "front" or 5' end of a
eukaryotic messenger RNA shortly after the start of transcription.
The 5' cap consists of a terminal 6-methylguanosine residue that is
linked through a 5'-5'-triphosphate bond to the first transcribed
nucleotide. Its presence is critical for recognition by the ribosome
and protection from RNases.
• Shortly after the start of transcription, the 5' end of the
mRNA being synthesized is bound by a cap-synthesizing complex
associated with RNA polymerase. This enzymatic complex
catalyzes the chemical reactions that are required for mRNA
capping. Synthesis proceeds as a multi-step biochemical reaction.
Splicing
Splicing is the process by which pre-mRNA is modified to
remove certain stretches of non-coding sequences called
introns; the stretches that remain include protein-coding
sequences and are called exons. Sometimes pre-mRNA
messages may be spliced in several different ways, allowing
a single gene to encode multiple proteins. This process is
called alternative splicing. Splicing is usually performed by
an RNA-protein complex called the spliceosome, but some
RNA molecules are also capable of catalyzing their own
splicing.
Editing
Polyadenylation
Polyadenylation is the covalent linkage of a polyadenylyl
moiety to a messenger RNA molecule. In eukaryotic
organisms, with the exception of histones, all messenger
RNA (mRNA) molecules are polyadenylated at the 3' end.
The poly (A) tail and the protein bound to it aid in protecting
mRNA from degradation by exonucleases. Polyadenylation
is also important for transcription termination, export of the
mRNA from the nucleus, and translation. mRNA can also be
polyadenylated in prokaryotic organisms, where poly(A)
tails act to facilitate, rather than impede, exonucleolytic
degradation.
Polyadenylation occurs during and immediately after
transcription of DNA into RNA. After transcription has been
terminated, the mRNA chain is cleaved through the action of an
endonuclease complex associated with RNA polymerase. After
the mRNA has been cleaved, around 250 adenosine residues are
added to the free 3' end at the cleavage site. This reaction is
catalyzed by polyadenylate polymerase. Just as in alternative
splicing, there can be more than one polyadenylation variant of
an mRNA.
Polyadenylation site mutations also occur. The primary RNA
transcript of a gene is cleaved at the poly-A addition site, and
100-200 A’s are added to the 3’ end of the RNA. If this site is
altered, an abnormally long and unstable mRNA results. Several
beta globin mutations alter this site: one example is AATAAA -
> AACAAA. Moderate anemia was result.
RNA polymerase III
RNA polymerase III
RNA polymerase III (Pol III) transcribes small non-coding RNAs,
including tRNAs, 5S rRNA, U6 snRNA, SRP RNA, and other
stable short RNAs such as ribonuclease P RNA.
Structure of eukaryotic RNA polymerase
RNA Polymerases I, II, and III contain 14, 12, and 17
subunits, respectively.
All three eukaryotic polymerases have five core subunits
that exhibit homology with the β, β’, αI, αII, and ω subunits of E.
coli RNA polymerase.
An identical ω-like subunit (RBP6) is used by all three
eukaryotic polymerases, while the same α-like subunits are used by
Pol I and III.
The three eukaryotic polymerases share four other
common subunits among themselves. The remaining
subunits are unique to each RNA polymerase. The
additional subunits found in Pol I and Pol III relative to
Pol II, are homologous to Pol II transcription factors.
Crystal structures of RNA polymerases I and II
provide an opportunity to understand the interactions
among the subunits and the molecular mechanism of
eukaryotic transcription in atomic detail.
Promoter for RNA polymerase – III
RNA pol-III transcribes small molecular weight
RNAs such as tRNAs, 5sRNAs, 7sKRNAs, 7sLRNAs,
U6sn RNAs, some ncRNAs and it also transcribes some
ADV, EBV and many eukaryotic viral genes.
The 5s rRNA and tRNA genes have promoters
within the coding region of the gene.
The promoter regions for 7S and U6sn RNAs,
more or less, look like RNA pol-II promoters, with little
differences.
Though the size of the genes is small ranging
from 160 to 400 bp, their promoters are well defined for
transcriptional initiation from their respective Start sites in
the promoters.
Initiation
Initiation: the construction of the polymerase complex on the
promoter. Pol III is unusual (compared to Pol II) requiring no
control sequences upstream of the gene, instead normally
relying on internal control sequences - sequences within the
transcribed section of the gene (although upstream sequences
are occasionally seen, e.g. U6 snRNA gene has an upstream
TATA box as seen in Pol II Promoters).
Class I
Typical stages in 5S rRNA (also termed class I) gene
initiation:
TFIIIA (Transcription Factor for polymerase III A) binds to
the intragenic (lying within the transcribed DNA sequence) 5S
rRNA control sequence, the C Block (also termed box C).
TFIIIA Serves as a platform that replaces the A and B
Blocks for positioning TFIIIC in an orientation with respect to
the start site of transcription that is equivalent to what is
observed for tRNA genes.
Once TFIIIC is bound to the TFIIIA-DNA complex the
assembly of TFIIIB proceeds as described for tRNA
transcription.
Class II
Typical stages in a tRNA (also termed class II) gene
initiation:
TFIIIC (Transcription Factor for polymerase III C) binds to
two intragenic (lying within the transcribed DNA sequence)
control sequences, the A and B Blocks (also termed box A and
box B).
TFIIIC acts as an assembly factor that positions TFIIIB to
bind to DNA at a site centered approximately 26 base pairs
upstream of the start site of transcription. TFIIIB (Transcription
Factor for polymerase III B), consists of three subunits: TBP
(TATA Binding Protein), the Pol II transcription factor TFIIB-
related protein, Brf1 (or Brf2 for transcription of a subset of Pol
III-transcribed genes in vertebrates) and Bdp1.
TFIIIB is the transcription factor that assembles Pol III at the
start site of transcription. Once TFIIIB is bound to DNA, TFIIIC
is no longer required. TFIIIB also plays an essential role in
promoter opening.
TFIIIB remains bound to DNA following initiation of
transcription by Pol III (unlike bacterial σ factors and most of the
basal transcription factors for Pol II transcription). This leads to
a high rate of transcriptional reinitiation of Pol III-transcribed
genes.
Class III
Typical stages in a U6 snRNA (also termed class III) gene
initiation (documented in vertebrates only):
SNAPc (SNRNA Activating Protein complex) (also termed
PBP and PTF) binds to the PSE (Proximal Sequence Element)
centered approximately 55 base pairs upstream of the start site
of transcription. This assembly is greatly stimulated by the Pol
II transcription factors Oct1 and STAF that bind to an
enhancer-like DSE (Distal Sequence Element) at least 200
base pairs upstream of the start site of transcription. These
factors and promoter elements are shared between Pol II and
Pol III transcription of snRNA genes.
SNAPc acts to assemble TFIIIB at a TATA box centered 26
base pairs upstream of the start site of transcription. It is the
presence of a TATA box that specifies that the snRNA gene is
transcribed by Pol III rather than Pol II.
The TFIIIB for U6 snRNA transcription contains a smaller
Brf1 paralogue, Brf2.
TFIIIB is the transcription factor that assembles Pol III at the
start site of transcription. Sequence conservation predicts that
TFIIIB containing Brf2 also plays a role in promoter opening.
Each of the internal sequence represents certain tRNA
domains, such as; A block representing D-arm and B block
representing TUCG loop respectively.
.
At the time of transcriptional initiation, a transcriptional factor
TF-C made up of six subunits recognizes the sequence boxes and
binds to them and positions the proteins in such a way one end of
the protein is found at the start site.
Then this protein guides the TF-B, which is made up of
several subunits, to be positioned at start site.
Then the RNA pol-III recognizes these proteins and binds to
them and binds tightly and initiates transcription at the pre
defined site.
Here the role of a promoter is to provide recognition sequence
modules for specific proteins to assemble in such a way; the
polymerase is properly positioned to initiate transcription exactly
at a pre-defined nucleotide, which is called start site.
If sequence motifs are not present, protein fails to bind
and RNA pol fails to associate with accessory proteins and
initiate transcription at specific site.
In these promoters there is sequence such as TATA box
for the binding of TBP, which acts as the positional factor.
This is what the promoter is and what it is meant for;
this is why promoter is required.
5sRNA genes:
Ribosomal RNAs, in eukaryotes consist of 28s, 18s,
5.8s and 5s RNAs.
The 28s, 18s and 5.8s rRNAs are synthesized as one
block from nucleolar organizer region of the DNA, and
the precursor 45S, larger than the final RNAs, is
processed into 28s, 18s, and 5.8s RNAs, but no 5s RNA
segment.
Gene for 5s RNA are located elsewhere in the
chromosomes, many times they are found just behind
telomeres.
The number of 5s RNA genes in a haploid genome can
vary from 200 to more than 1200, and all of them are
tandemly repeated in the cluster and each of them are
separated by non transcribing spacer.
During transcriptional initiation, TF III A first
recognizes the C box and binds, then TF-III-B containing
TBP binds to the promoter using TF-III A and it positions
at start site.
Then the RNA-pol-III complex assembles at the start
region and initiates transcription at the predefined site.
Again the role of internal promoters is to position the
transcriptional factors and ultimately the RNA-pol so as to initiate
at specified site.
5s RNA expression differs in Oocyte and somatic tissues.
Transcription factor TF III A, 40 KD proteins is produced in
Oocyte specific manner.
This protein binding to internal site of the 5s gene activates the
gene expression by facilitating the assembly of TF III-C and B and
finally RNA pol-III.
At a late stage of oogenesis, enormous quantities of 5sRNAs are
produced, and the TF-III A binds to 5s RNA, thus all TF III-As get
consumed and none of the factors are available for the activation of
Oocyte specific 5sRNA gene.
Termination
Polymerase III terminates transcription at small polyTs stretch. In
Eukaryotes, a hairpin loop is not required, as it is in prokaryotes
Processing
tRNA Synthesis & Processing
1. tRNA is transcribed by RNA polymerase III. The
transcription product, the pre-tRNA, contains additional RNA
sequences at both the 5’ and 3’-ends. These additional
sequences are removed from the transcript during processing.
The additional nucleotides at the 5’-end are removed by an
unusual RNA containing enzyme called ribonuclease P (RNase
P).
2. Some tRNA precursors contain an intron located in the
anticodon arm. These introns are spliced out during processing
of the tRNA.
3. All mature tRNAs contain the trinucleotide CCA at their 3’-
end. These three bases are not coded for by the tRNA gene.
Instead, these nucleotides are added during processing of the
pre-tRNA transcript. The enzyme responsible for the addition of
the CCA-end is tRNA nucleotidyl transferase and the reaction
proceeds according to the following scheme:
tRNA +CTP --> tRNA-C + PPi (pyrophosphate)
tRNA-C +CTP --> tRNA-C-C + PPi
tRNA-C-C +ATP --> tRNA-C-C-A + PPi
4. Mature tRNAs can contain up to 10% bases other than the
usual adenine (A), guanine (G), cytidine (C) and uracil (U).
These base modifications are introduced into the tRNA at the
final processing step. The biological function of most of the
modified bases is uncertain and the translation process seems
normal in mutants lacking the enzymes responsible for
modifying the bases.
α-Amanitin and actinomycin D are commonly
used inhibitors of transcription. α-Amanitin
binds to the largest subunits of RNA
polymerase II (RNAP II) and RNAP III, with
RNAP II being the most sensitive. As a
consequence, the incorporation of new
ribonucleotides into the nascent RNA chains
is blocked
Rifamycins, macrocyclic antibiotics produced
by Streptomyces mediterranei, inhibit the
bacterial RNA polymerase, by binding to the
beta subunit, which is one of the five
subunits of the enzyme: They have little
action on the human RNA polymerase. This
group of antibiotics includes rifampicin,
rifabutin and rifamycine SV.
INHIBITORS OF TRANSCRIPTION
Rifampin
Rifampin, also called rifampicin, has a
bactericidal activity against a wide range of
microorganisms, of which Mycobacterium
tuberculosis and Mycobacterium lepræ as well
as staphylococci, streptococci, Neisseria,
Listeria monocytogenes, Brucella…
It is used as antituberculous drug, always
combined to two or three other drugs to avoid
the emergence of resistance and as anti-leprous
drug. Its other clinical uses are brucellosis and
the prophylaxis of meningococcal meningitis.
Rifampicin (Rifadin*, Rimactan*) is marketed
alone and in combination with isoniazid
(Rifinah*) and with isoniazid and pyrazinamid
(Rifater*).
Rifabutin
Rifabutin has an antibacterial activity quite
similar to that of rifampin, it is active against
mycobacteria such as Mycobacterium
tuberculosis and Mycobacterium avium complex.
It is also active against several gram-positive
bacteria.
Rifabutin (Mycobutin*) is used for the curative
treatment of multidrug-resistant tuberculosis and
for the prophylactic treatment of Mycobacterium
avium complex infection in immunocompromised
patients. Rifabutin is a less potent microsomal
enzyme inducer than rifampin and can be
preferred in patients taking other drugs.
Rifampin and rifabutin can elicit a rise in hepatic
transaminases and thrombocytopenia and
neutropenia. They give an orange color to the
urine. Rifabutin can cause uveitis.
Rifamycine
Rifamycine S.V is used in the form of
ophthalmic solution.
Rifapentine
Rifapentine is a rifampin analog used
in certain countries for tuberculosis
therapy.
V. Magendira ManiAssistant Professor, PG & Research Department of Biochemistry,Islamiah College (Autonomous),Vaniyambadi,Vellore District – 6357512,Tamilnadu, [email protected] ; vinayagam [email protected]
https://tvuni.academia.edu/mvinayagam
