ROLES OF RNA IN CELLS
 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.

All 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.

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 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.

1. 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.
2. 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

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.

 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.

TRANSCRIPTION IN
PROKARYOTES
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.

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).

INITIATION

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.

ELONGATION
 During elongation, RNA polymerase binds to about 30 base
pairs of DNA (each complete turn 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.

TERMINATION
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.

SUMMARY: PROKARYOTIC TRANSCRIPTION
1.Transcription is a selective process; only certain
parts of the DNA are transcribed.
2.RNA is transcribed from single-stranded DNA.
Normally, only one of the two DNA strands, the
template strand, is copied into RNA.
3.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.

5.RNA molecules are antiparallel and complementary to the
DNA template strand.
6.Transcription is always in the 5’-3’ direction, which means
that the RNA molecule grows at the 3’ end.
7.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.
8.The core enzyme of RNA polymerase requires a sigma
factor in order to bind to a promoter and initiate
transcription.
9.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.
10.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
TRANSCRIPTION yield a
IN EUKARYOTES functional
promoter.

 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.

TERMINATION
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).

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.

SUMMARY: EUKARYOTIC TRANSCRIPTION
 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.

 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.

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.

 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:
o Regulation of nuclear
export
o Prevention of
degradation
by exonucleases
o Promotion of translation
o 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.

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
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.

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.

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.

: 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:
o removal of the leader sequence at the 5’ end
o replacement of two nucleotides at the 3’ end by
the sequence CCA (with which all mature tRNA
molecules terminate)
o chemical modification of certain bases
o 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.

Long double-stranded RNAs  Upon introduction,
(dsRNA) occur naturally in cells. 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
by an RNase
III-like enzyme called
Dicer.

 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
.......geneticsvideosRNAi.wmv
silencing.

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