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DNA 
TRANSCRIPTION
 RNA molecules can function as biological 
catalysts and may have been the first carriers 
of genetic information.
 RNA is a polymer, consisting of nucleotides 
joined together by phosphodiester bonds. 
 Each RNA nucleotide consists of a ribose 
sugar, a phosphate, and a base. RNA contains 
the base uracil
 It is usually single stranded, which allows it to 
form secondary structures.
ROLES OF RNA IN CELLSROLES OF RNA IN CELLS
All cellular RNA types are transcribed from DNAAll cellular RNA types are transcribed from DNA
RNA are synthesized that are complementary and 
antiparallel to the DNA template strand.
In most organisms, each gene is transcribed from a 
single DNA strand, but different genes maybe 
transcribed from one or the other of the two DNA 
strands.
Polymerization of ribonucleotides by 
RNA polymerase during transcription.
 The ribonucleotide to be added at the 
3’ end of a growing RNA strand is 
specified by base pairing between the 
next base in the template DNA strand 
and the complementary incoming 
ribonucleoside triphosphate (rNTP). 
A phosphodiester bond is formed 
when RNA polymerase catalyzes a 
reaction between the 3’ O of the 
growing strand and theα phosphate of 
a correctly base-paired rNTP. RNA 
strands always are synthesized in the 
5’-3’ direction and are opposite in 
polarity to their template DNA strands.
The genetic code is a system of purines and pyrimidines used to 
send messages from the genome to the ribosomes to direct 
protein synthesis. With a non-overlapping code, the reading 
frame advances three nucleotides at a time, and a mRNA 
segment is therefore read as three successive triplets, coding for 
amino acids. 
•The genetic code are triplet , 
commaless, non-overlapping codons 
present in the nucleotide sequence of 
mRNA, as read in the 5’-3’ direction. 
•Each codon specifies either an amino 
acid or a stop signal. 
•There are 64 possible codons in mRNA, 
61 code for amino acids, hence, 
degenerate and wobbling). 
•TAA, TAG and TGA are the stop codons 
which do not have a corresponding 
tRNA. 
•The genetic code is universal. 
THE TRANSCRIPTION APPARATUSTHE TRANSCRIPTION APPARATUS
In bacterial RNA polymerase, the core enzyme consists of 4 catalytic 
subunits: 2 copies of alpha (α), a single copy of beta (β), and single 
copy of beta prime (β’). A 5th unit (ω) has been identified recently. (a) 
The regulatory subunit known as the sigma (σ) factor joins the core to 
form the holoenzyme, which is capable of binding to a promoter and 
initiating transcription. 
(b) The molecular model shows RNA polymerase (shown in yellow) 
binding DNA.
• The core enzyme catalyzes the elongation of the RNA molecule by 
the addition of RNA nucleotides. 
• α2: The two α subunits assemble the enzyme and bind regulatory 
factors. 
• ß: this has the polymerase activity which includes chain initiation and 
elongation.
• ß': binds to DNA (nonspecifically). 
• ω: restores denatured RNA polymerase to its functional form in vitro. It 
has been observed to offer a protective/chaperone function to the β' 
subunit. 
• Once bound, the sigma factor increases RNA polymerase specificity 
for certain promoter regions, depending on the specific σ factor. That 
way, transcription is initiated at the right region. 
• When not in use, RNA polymerase binds to low-affinity sites to allow 
rapid exchange for an active promoter site when one opens. RNA 
polymerase holoenzyme, therefore, does not freely float around in the 
cell when not in use. 
• Eukaryotes have several types of RNA polymerases, characterized 
by the type of RNA they synthesize:
• RNA polymerase I synthesizes a pre-rRNA 45S, which matures 
into 28S, 18S and 5.8S rRNAs which will form the major RNA 
sections of the ribosome.
• RNA polymerase II synthesizes precursors of mRNAs and 
most snRNA and microRNAs. This is the most studied type, and 
due to the high level of control required over transcription, a range 
of transcription factors are required for its binding to promoters.
• RNA polymerase III synthesizes tRNAs, rRNA 5S and other small 
RNAs found in the nucleus and cytosol.
• RNA polymerase IV synthesizes siRNA in plants.
• RNA polymerase V synthesizes RNAs involved in siRNA-
directed heterochromatin formation in plants.
• There are RNA polymerase types in mitochondria and chloroplasts.
• There are RNA-dependent RNA polymerases involved in RNA 
interference.
Rifampicin 
inhibits 
prokaryotic RNA 
polymerases;
α-Amanitin 
eukaryotic RNA 
polymerase II
3 kinds of RNA polymerases
A transcription unit is a piece of DNA that encodes 
an RNA molecule and the sequences necessary for 
its proper transcription. Each transcription unit 
includes a promoter, an RNA-coding region, and a 
terminator.
• A promoter is a DNA sequence that is adjacent to a gene and 
required for transcription. 
• Promoters contain short consensus sequences that are important 
in the initiation of transcription.
• Consensus sequence comprises the most commonly encountered 
nucleotides found at a specific location.
• In bacterial promoters, consensus sequences are found upstream 
of the start site, approximately at positions 10 & 35. 
Transcription: An Overview
• In all species, transcription begins with the binding of 
the RNA polymerase complex (or holoenzyme) to a 
special DNA sequence at the beginning of the gene known 
as the promoter. 
• Activation of the RNA polymerase complex enables 
transcription initiation, and this is followed by elongation of 
the transcript. 
• In turn, transcript elongation leads to clearing of the 
promoter, and the transcription process can begin yet 
again. 
• Transcription can thus be regulated at two levels: the 
promoter level (cis regulation) and the polymerase level 
(trans regulation). 
• These elements differ among bacteria and eukaryotes.
• A promoter is a DNA sequence that is adjacent to a gene and 
required for transcription. 
• Promoters contain short consensus sequences that are important 
in the initiation of transcription.
• Consensus sequence comprises the most commonly encountered 
nucleotides found at a specific location.
• In bacterial promoters, consensus sequences are found upstream 
of the start site, approximately at positions 10 & 35. 
In prokaryotic DNA, several 
protein-coding genes 
commonly are clustered 
into a functional region, an 
operon, which is 
transcribed from a single 
promoter into one mRNA 
encoding multiple proteins 
with related functions. 
Translation of a bacterial 
mRNA can begin before 
synthesis of the mRNA is 
complete.
TRANSCRIPTION 
IN PROKARYOTES
TRANSCRIPTION 
IN PROKARYOTES
During initiation of transcription, 
RNA polymerase forms a 
transcription bubble and begins 
polymerization of rNTPs at the start 
site, which is located within the 
promoter region. RNA polymerase 
moves along the template strand of 
the DNA in the 3’- 5’direction, and 
the RNA molecule grows in the 5’- 
3’ direction. Once a DNA region has 
been transcribed, the separated 
strands reassociate into a double 
helix, displacing the nascent RNA 
except at its 3’ end. The 5’ end of 
the RNA strand exits the RNA 
polymerase through a channel in 
the enzyme. Termination occurs 
when the polymerase encounters a 
termination sequence (stop site).
INITIATIONINITIATION
Transcription is initiated at the start site, which, in bacterial 
cells, is set by the binding of RNA polymerase to the consensus 
sequences of the promoter. Transcription takes place within the 
transcription bubble. DNA is unwound ahead of the bubble and 
rewound behind it.
 During elongation, RNA polymerase binds to about 30 base pairs of 
DNA (each completeturn of the DNA double helix is about 10 base 
pairs). 
 At any given time, about 18 base pairs of DNA are unwound, and 
the most recently synthesized RNA is still hydrogen-bonded to the 
DNA, forming a short RNA-DNA hybrid. 
 This hybrid is probably about 12 base pairs long, even shorter. The 
total length of growing RNA bound to the enzyme and/or DNA is 
about 25 nucleotides. 
ELONGATIONELONGATION
Transcription ends after RNA 
polymerase
transcribes 2 types of 
terminator sequences: in rho-
independent
termination, a GC-rich 
sequence followed by several U 
residues forms a "brake" that 
will help release the RNA 
polymerase from the template. 
In rho-dependent termination, 
binding of rho to the mRNA 
releases it from the template.
TERMINATIONTERMINATION
• Transcription is a selective process; only certain parts of 
the DNA are transcribed. 
• RNA is transcribed from single-stranded DNA. Normally, 
only one of the two DNA strands, the template strand, is 
copied into RNA.
• Ribonucleoside triphosphates (RNTPs), are used as the 
substrates in RNA synthesis. Two phosphates are cleaved 
from an RNTP, and the resulting nucleotide is joined to the 
3’OH group of the growing RNA strand.
SUMMARY: PROKARYOTIC TRANSCRIPTIONSUMMARY: PROKARYOTIC TRANSCRIPTION
• RNA molecules are antiparallel and complementary to the DNA 
template strand. 
• Transcription is always in the 5’-3’ direction, which means that 
the RNA molecule grows at the 3’ end. 
• Transcription depends on RNA polymerase- a complex, 
multimeric enzyme which consists of a core enzyme capable of 
synthesizing RNA, and other subunits that may join transiently 
to perform additional functions.
• The core enzyme of RNA polymerase requires a sigma factor in 
order to bind to a promoter and initiate transcription.
• Promoters contain short sequences crucial in the binding of 
RNA polymerase to DNA; these consensus sequences are 
interspersed with nucleotides that play no known role in 
transcription.
• RNA polymerase binds to DNA at a promoter, begins 
transcribing at the start site of the gene, and ends transcription 
after a terminator has been transcribed.
• The consensus 
sequences in 
promoters of 
three eukaryotic 
genes illustrate 
the principle 
that different 
sequences can 
be mixed and 
matched to yield 
a functional 
promoter.
TRANSCRIPTION IN 
EUKARYOTES
TRANSCRIPTION IN 
EUKARYOTES
Eukaryotic nuclear genes have 3 classes of promoters 
which are specific for the 3 types of RNA polymerases: 
RNA polymerase I transcribes the rRNA precursor 
molecules. 
RNA polymerase I promoters have two key components: (1) 
the core element, which surrounds the start site and is 
sufficient to initiate transcription, and (2) the upstream 
control sequence, which increases the efficiency of the core 
promoter. 
The basal transcription apparatus assembles at 
RNA polymerase I promoters.
RNA polymerase II produces most mRNAs and snRNAs. 
The promoters of genes transcribed by RNA polymerase II 
consist of a core promoter and a regulatory promoter that 
contain consensus sequences. 
Not all the consensus sequences shown are found in all 
promoters.
The typical promoter for RNA polymerase II has a 
short initiator sequence, consisting mostly of 
pyrimidines and usually a TATA box about 25 bases 
upstream from the start point. 
This type of promoter (with or without the TATA box) is 
often called a polymerase II core promoter, because 
for most genes a variety of upstream control elements 
also play important roles in the initiation of 
transcription.
RNA polymerase III is 
responsible for the 
production of pre-tRNAs, 
5SrRNA and other small 
RNAs. 
RNA polymerase III 
recognizes several 
different types of 
promoters. 
OCT and PSE are 
consensus sequences 
that may also be present 
in RNA polymerase II 
promoters.
The promoters for RNA polymerase III vary in structure 
but the ones for tRNA genes and 5S rRNA genes are 
located entirely downstream of the startpoint, within the 
transcribed sequence. 
In tRNA genes, about 30-60 base-pairs of DNA separate 
promoter elements; in 5S rRNA genes, about 10-30 base-
pairs promoter elements
General transcription 
factors and the 
polymerase undergo a 
pattern of sequential 
binding to initiate 
transcription of nuclear 
genes. 
(1) TFIID binds to the 
TATA box followed by 
(2) the binding of TFIIA 
and TFIIB. 
(3) The resulting 
complex is now bound 
by the polymerase, to 
which TFIIF has already 
attached. 
● (4) The initiation 
complex is 
completed by the 
addition of TFIIE, 
and TFIIH. TFIIH 
helicase activity 
and its associated 
kinase complex 
referred to as 
TFIIK 
phosphorylates the 
C-terminal domain 
of RNA polymerase 
largest subunit. 
(5) After its ATP-
dependent 
phosphorylation, 
the polymerase 
can initiate 
transcription at the 
startpoint. 
The TATA-binding protein (TBP) is a subunit of the 
TFIID and plays a role in the activity of both RNA 
polymerase I and III transcription. 
TBP is also essential for transcription of TATA-less genes. 
TBP differs from most DNA-binding proteins in that it 
interacts with the minor groove of DNA, rather than the 
major groove and imparts a sharp bend to the DNA. 
TBP has been highly conserved during evolution. 
When TBP is bound to DNA, other transcription-factor 
proteins can interact with the convex surface of the TBP 
saddle. 
TBP is required for transcription initiation on all types of 
eukaryotic promoters. 
In many of the genes transcribed by RNA polymerase 
II, transcription can end at multiple sites located within a 
span of hundreds or thousands of base pairs. 
Termination is coupled to cleavage, which is carried out 
by a termination factor that associates with RNA 
polymerase I and III.
This complex may suppress termination until the 
consensus sequence that marks the cleavage site is 
encountered. 
mRNA is cleaved by the complex 10 to 35 base-pairs 
downstream of a AAUAAA sequence (which acts as a 
poly-A tail addition signal). 
TERMINATIONTERMINATION
Unlike rho, which binds to the newly transcribed 
RNA molecule, the termination factor for RNA 
polymerase I binds to a DNA sequence 
downstream of the termination site. 
RNA polymerase III transcribes a terminator 
sequence that produces a string of U’s in the RNA 
molecule, like that produced by the rho-
independent terminators of bacteria. 
Unlike rho-independent terminators in bacterial 
cells, RNA polymerase III does not require that a 
hairpin structure precede the string of U’s. 
Several types of DNA sequences take part in the initiation of 
transcription in eukaryotic cells. These promoter sequences 
generally serve as the binding sites for proteins that interact 
with RNA polymerase and influence the initiation of 
transcription.
 Promoters are adjacent to or within the RNA coding region 
and are relatively fixed with regard to the start site of 
transcription. 
Promoters consist of a core promoter located adjacent to the 
gene and a regulatory promoter located farther upstream.
Other sequences, called enhancers, are distant from the 
gene and function independently of position and direction. 
Enhancers stimulate transcription.
SUMMARY: EUKARYOTIC TRANSCRIPTIONSUMMARY: EUKARYOTIC TRANSCRIPTION
General transcription factors bind to the core promoter 
near the start site and, with RNA polymerase, assemble into 
a basal transcription apparatus. 
The TATA-binding protein (TBP) is a critical transcription 
factor that positions the active site of RNA polymerase over 
the start site.
Transcriptional activator proteins bind to sequences in the 
regulatory promoter and enhancers and affect transcription 
by interacting with the basal transcription apparatus.Proteins binding to enhancers interact with the basal 
transcription apparatus by causing the DNA between the 
promoter and the enhancer to loop out, bringing the 
enhancer into close proximity to the promoter. 
The three RNA polymerases found in eukaryotic cells use 
different mechanisms of termination.
Several types of DNA sequences take part in the initiation of 
transcription in eukaryotic cells. These promoter sequences 
generally serve as the binding sites for proteins that interact 
with RNA polymerase and influence the initiation of 
transcription.
 Promoters are adjacent to or within the RNA coding region 
and are relatively fixed with regard to the start site of 
transcription. 
Promoters consist of a core promoter located adjacent to the 
gene and a regulatory promoter located farther upstream.
Other sequences, called enhancers, are distant from the 
gene and function independently of position and direction. 
Enhancers stimulate transcription.
SUMMARY: EUKARYOTIC TRANSCRIPTIONSUMMARY: EUKARYOTIC TRANSCRIPTION
● Transcription of eukaryotic pre-mRNAs often proceeds beyond the 3’ end of 
the mature mRNA. An AAUAAA sequence located slightly upstream from the 
proper 3’ end then signals that the RNA chain should be cleaved about 10-35 
nucleotides downstream from the signal site, followed by addition of a poly-A 
tail catalyzed by poly(A) polymerase.
mRNA 
processing
mRNA 
processing
A 5’ “cap” (a guanosine 
nucleotide methylated at the 7th 
position) is joined to the 1st 
nucleotide in an unusual 5’ -5’ 
linkage.
The 5' cap has 4 main functions:
Regulation of nuclear export
Prevention of degradation 
by exonucleases
Promotion of translation
Promotion of 5' proximal intron 
excision
During the capping process, the 
first two nucleotides of the 
message may also become 
methylated.
 The poly(A) tail is important for the nuclear export, 
translation and stability of mRNA. The tail is shortened 
over time and when it is short enough, the mRNA is 
enzymatically degraded.
 In addition to the 5’ cap and poly-A tail, mRNA in 
eukaryotes is first made as heterogeneous nuclear 
mRNA (or pre-mRNA), and then processed into mature 
mRNA through the splicing out of introns.
Restriction 
enzyme 
analysis has 
revealed the 
presence of 
introns in 
eukaryotic 
DNA.
Hybridization of a 
eukaryotic mRNA 
molecule with a 
gene which has one 
intron will produce 
two single-stranded 
DNA loops where 
the mRNA has 
hybridized to the 
DNA template 
strand plus an 
obvious double-
stranded DNA loop. 
The double-
stranded DNA loop 
represents the 
intron, which 
contains sequences 
that do not appear 
in the final mRNA. 
Alternative splicing results in alternate forms of 
mRNA and proteins.
The ≈75-kb fibronectin gene (top) contains multiple exons. The 
EIIIB and EIIIA exons (green) encode binding domains for specific 
proteins on the surface of fibroblasts. The fibronectin mRNA 
produced in fibroblasts includes the EIIIA and EIIIB exons, 
whereas these exons are spliced out of fibronectin mRNA in 
hepatocytes. In this diagram, introns (black lines) are not drawn to 
scale; most of them are much longer than any of the exons.
Distinct isoforms of individual domains of multidomain proteins found 
in higher eukaryotes often are expressed in specific cell types as the 
result of alternative splicing of exons
Spliceosomes remove introns from pre-mRNA. The 
spliceosome is an RNA-protein complex that splices intron-
containing pre-mRNA in the eukaryotic nucleus. 
http://highered.mcgraw-hill.com/olc/dl/120077/bio30.swf
In a stepwise 
fashion, the pre-
mRNA assembles 
with the U1 
snRNP, U2 
snRNP, and 
U4/U6 and U5 
snRNPs (along 
with some non-
snRNP splicing 
factors), forming a 
mature 
spliceosome.
The pre-mRNA is then 
cleaved at the 5’ splice 
site and the newly 
released 5’ end is linked 
to an adenine (A) 
nucleotide located at the 
branch-point sequence, 
creating a looped lariat 
structure. Next the 3’ 
splice site is cleaved 
and the two ends of the 
exon are joined 
together, releasing the 
intron for subsequent 
degradation.
Clinical Significance: 
Alternative and Aberrant Splicing
Introns protect the genetic makeup of an organism from genetic 
damage by outside influences such as chemical or radiation, and 
increase the genetic diversity of the genome without increasing the 
overall number of genes. 
Abnormalities in the splicing process can lead to various disease 
states. Many defects in the β-globin genes are known to exist 
leading to β-thalassemias. Some of these defects are caused by 
mutations in the sequences of the gene required for intron 
recognition and, therefore, result in abnormal processing of the β-
globin primary transcript.
Patients suffering from a number of different connective tissue 
diseases exhibit humoral auto-antibodies that recognize cellular 
RNA-protein complexes. Patients suffering from systemic lupus 
erythematosis have auto-antibodies that recognize the U1 RNA of 
the spliceosome.
Ribosomal RNA processing involves cleavage of multiple 
rRNAs from a common precursor. 
The eukaryotic transcription unit that includes the genes for 
the three largest rRNAs occurs in multiple copies and 
arranged in tandem arrays with non-transcribed spacers 
separate the units. 
Each transcription unit includes the genes for the three 
rRNAs and transcribed spacer regions. 
The transcription unit is transcribed by RNA polymerase I 
into a single long transcript (pre-rRNA) with a 
sedimentation coefficient of about 45S. 
RNA processing yields mature rRNA molecules. 
RNA cleavage actually occurs in a series of steps which 
varies in order with the species and cell type but the final 
products are always the same three types of rRNA 
molecules.
Transfer RNA processing: every tRNA gene is transcribed 
as a precursor that must be processed into a mature tRNA 
molecule by the removal, addition and chemical 
modification of nucleotides. 
Processing for some tRNA involves: 
removal of the leader sequence at the 5’ end 
replacement of two nucleotides at the 3’ end by the 
sequence CCA (with which all mature tRNA molecules 
terminate) 
chemical modification of certain bases 
excision of an intron
The mature tRNA is often diagrammed as a flattened 
cloverleaf which clearly shows the base pairing between 
self-complementary stretches in the molecule.
Small RNAs and Post-Transcrip-tional Regulation
Upon introduction, the 
long dsRNAs with 
complementary 
sequence of a part of the 
target gene, enter a 
cellular pathway that is 
commonly referred to as 
the RNA interference 
(RNAi) pathway
The dsRNAs get 
processed into 20-25 
nucleotide small 
interfering RNAs 
(siRNAs) by an RNase 
III-like enzyme called 
Dicer. 
Long double-stranded RNAs 
(dsRNA) occur naturally in cells. 
The siRNAs assemble into 
endoribonuclease containing 
complexes known as RNA-
induced silencing complexes 
(RISCs), unwinding in the 
process.
Activated RISC then binds to 
complementary transcript by 
base pairing interactions 
between the siRNA antisense 
strand and the mRNA.
The bound mRNA is cleaved 
and sequence specific 
degradation of mRNA results 
in gene silencing.
...\..\..\genetics\videos\RNAi.wmv
MicroRNAs ("miRNAs") 
are single-stranded RNA 
molecules containing about 
22 nucleotides and thus 
about the same size as 
siRNAs. 
These are generated by the 
cleavage of larger precursors 
using Dicer. 
They function as post-
transcriptional regulators of 
gene expression.
They act by either destroying 
or inhibiting translation of 
several mRNAs, usually by 
binding to a region of 
complementary sequence in 
the 3'-UTR region of the 
mRNA.http://www.nature.com/ng/supplements/micrornas/video.html
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