Mrs.

OFELIA SOLANO SALUDAR

Department of Natural Sciences
University of St. La Salle
Bacolod City
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 RNA

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 3-
letter 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.
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 four subunits:

two copies of alpha, a single copy of beta, and single copy of beta prime.
The core enzyme catalyzes the elongation of the RNA molecule by the
addition of RNA nucleotides. (a) The sigma 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.
 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 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.
 TERMINATIO
N ends after
Transcription
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.
4. 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.
5. Transcription depends on RNA polymerase- a complex,
multimeric enzyme. RNA polymerase consists of a core
enzyme, which is capable of synthesizing RNA, and other
subunits that may join transiently to perform additional
functions.
6. The core enzyme of RNA polymerase requires a sigma factor
in order to bind to a promoter and initiate transcription.
7. 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.
8. 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.
 TRANSCRIPTION IN EUKARYOTES

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 consensus
sequences in
promoters of
three
eukaryotic
genes illustrate
the principle
that
different
sequences can
be mixed and
matched to
yield a
functional
promoter.
Eukaryotic nuclear genes have 3 classes of promoters which are
specific for the 3 types of RNA polymerases:
1. RNA polymerase I: 
 Localized to the nucleolus, it transcribes the rRNA
precursor molecules.
 The promoter for RNA polymerase I has two components:
a core promoter (surrounding the start point), and an
upstream control element.
 After the binding of appropriate transcription factors to
both sites, RNA polymerase I binds to the core promoter.
2. RNA polymerase II: 
 This produces most mRNAs and snRNAs.
 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.
 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,
TFIIJ, and TFIIH.

(5) After an
activation step
requiring ATP-
dependent
phosphorylation of
the RNA
polymerase
molecule, 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.
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.
 To give the mRNA
stability, a  5’ “cap” (a
guanosine nucleotide
methylated at the 7th
position) is joined to the
1st nucleotide in an
unusual  5’ -5’ linkage.
During the capping
process, the first two
nucleotides of the
message may also
become methylated.
 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.
 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.
 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 sequences generally serve
as the binding sites for proteins that interact with RNA polymerase
and influence the initiation of transcription.
 Sequences that affect transcription, called 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.
EUKARYOTIC RNA POLYMERASES
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 III
recognizes several
different types of
promoters. OCT and
PSE are
consensus sequences
that may also be
present in RNA
polymerase II
promoters.
 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.
 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 (dsRNA)  Upon introduction, the
occur naturally in cells. 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.
  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