Transcription - prokaryotic & Eukaryotic, transcription regulatory elements, inhibitors of Transcription

1. OVERVIEW OF
TRANSCRIPTION
V. Magendira Mani
Assistant Professor,
PG & Research Department of
Biochemistry,
Islamiah College (Autonomous),
Vaniyambadi,
Vellore District – 6357512,
Tamilnadu, India.
magendiramani@rediffmail.com
Also available at
https://tvuni.academia.edu/mvinayagam

2. 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. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. Activators
Activators 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.
Repressors
Repressors 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.

8. Enhancer
In 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.

9. 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.

10. 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.

11. Promoter
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.
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.

12. 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.

13. 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.

14.  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

15. PROKARYOTIC
TRANSCRIPTION

16. 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.

17. 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.

18. 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.

19. 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.

20. 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

21. 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.

22. -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.

23. 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.

25. 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.

26. 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.

27. 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.

29. 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.

30. 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

31. EUKARYOTIC
TRANSCRIPTION

32. 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.

33. Eukaryotes have three nuclear RNA polymerases, each
with distinct roles and properties
Name 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.

34. 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.

35. 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.

37. 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.

39. 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.

40. 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.

42. 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.

43. 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.

44. 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.

45. 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.

47. 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.

49. 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.

50. 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.

51. 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.

52. 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.

53. 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.

54. 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.

55. 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.

56. 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).

57. 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).

58. 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.

60. 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.

62. 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.
.

63. 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.

64. 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.

65. 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.

66. 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

67. 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.

69. 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.

70. 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

71. 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*).

72. 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.

73. 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.

74. V. Magendira Mani
Assistant Professor,
PG & Research Department of Biochemistry,
Islamiah College (Autonomous),
Vaniyambadi,
Vellore District – 6357512,
Tamilnadu, India.
magendiramani@rediffmail.com ;
vinayagam magendiramani@academia.edu
https://tvuni.academia.edu/mvinayagam