SBT 211

RNA Synthesis, Processing,

& Modification
DNA transcription is a process that involves the
transcribing of genetic information from DNA to
RNA. The transcribed DNA message is used to
produce proteins.
The synthesis of an RNA molecule from DNA is a
complex process involving one of the group of RNA
polymerase enzymes and a number of associated
proteins.
The general steps required to synthesize the
primary transcript are initiation, elongation, and
termination.
The RNA molecules synthesized in mammalian
cells are made as precursor molecules that have to
be processed into mature, active RNA.
Types of RNA
All eukaryotic cells have four major classes of RNA:
ribosomal RNA (rRNA), messenger RNA (mRNA),
transfer RNA (tRNA), and small nuclear RNA
(snRNA). The first three are involved in protein
synthesis, and snRNA is involved in mRNA splicing.
Prokaryotic DNA dependent RNA
Polymerase (RNAP)
Bacterial cells have a single RNA polymerase that
transcribes DNA to generate all of the different
types of RNA (mRNA, rRNA, and tRNA).
Using DNA as a template, this enzyme polymerizes
ribonucleoside triphosphates (RNA nucleotides).
The complete RNA polymerase enzyme of E. coli
—the holoenzyme—is composed of a core enzyme
and a sigma factor.
An approximately 400 kDa core complex
consisting of two identical α subunits, similar but
not identical β and β′ subunits, and an ω subunit.
Beta is thought to be the catalytic subunit
RNAP, a metalloenzyme, also contains two zinc
molecules. The core RNA polymerase associates
with a specific protein factor (the sigma [σ] factor)
that helps the core enzyme recognize and bind to
the specific deoxynucleotide sequence of the
promoter region to form the preinitiation complex
(PIC)
Bacteria contain multiple σ factors, each of which
acts as a regulatory protein that modifies the
promoter recognition specificity of the RNA
polymerase. The major σ factor is σ70 , a
designation related to its molecular weight of
70,000 Daltons.
Following the initiation of transcription, the sigma
factor disassociates from the core enzyme.
Mammalian Cells Possess Three Distinct
Nuclear DNA-Dependent RNA Polymerases
In contrast to prokaryotes, eukaryotic cells have
three RNA polymerases.
◦ Polymerase I produces most of the rRNAs,
◦ polymerase II produces mRNA, and
◦ polymerase III produces small RNAs, such as tRNA and
5S rRNA.
All of these RNA polymerases have the same
mechanism of action. However, they recognize
different types of promoters.
They all have two large subunits and a number of
smaller subunits—as many as 14 in the case of
RNA pol III.
Actinomycin blocks the translocation of RNA
polymerase during transcription
Bacterial Transcription
Initiation
RNA polymerase (RNAP) binds to one of several
specificity factors, σ, to form a holoenzyme. In this
form, it can recognize and bind to specific promoter
regions in the DNA.
The DNA region that RNA polymerase associates with
immediately before beginning transcription is known as
the promoter.
The base pair where transcription initiates is called the
transcription-initiation site, or start site.
By convention, the transcription-initiation site in the
DNA sequence of a transcription unit is usually
numbered +1. Base pairs extending in the direction of
transcription (downstream) are assigned positive
numbers and those extending in the opposite direction
(upstream) are assigned negative numbers.
Promoters
RNA polymerase initiates transcription of most genes at
a unique position (a single base) in the template DNA
lying upstream of the coding sequence.
Surrounding a point in prokaryotic promoters about ten
nucleotides (-10) before the rst transcribed base is a
consensus sequence—TATAAT. This sequence is
known as a Pribnow box after one of its discoverers.
(A consensus sequence is the sequence most
commonly found in a given region when many genes
are examined.)
The nucleotides in the Pribnow box are mostly
adenines and thymines, so the region is primarily held
together by only two hydrogen bonds per base pair.
There is another region with similar sequences among
promoters centred near -35 and referred to as the -35
sequence. The consensus sequence at -35 is TTGTCA.
Promoter of the Escherichia coli ribosomal RNA gene, rrnB.
Note the -10 and -35 sequences and the upstream element.
The rst base transcribed (the transcriptional start site) is
noted (+1), as well as the upstream, downstream, and
transcription directions. (Data from W. Ross, et al., 1993.
Science 262:1407.)

The template (anticoding) strand of DNA is complementary

to both the coding strand and the transcribed RNA.
The sigma factor recognizes both the -35 and the
-10 sequences.
This initiation complex is initially referred to as a
closed complex because the DNA has not melted,
which is the next step in transcription initiation.
The DNA is unwound and becomes single-stranded
("open") in the vicinity of the initiation site (defined
as +1). This holoenzyme/unwound-DNA structure
is called the open complex.
After the transcription of 5–10 bases, the sigma
factor is released
About seventeen base pairs of DNA are opened,
and as transcription proceeds, about twelve bases
of RNA form a DNA-RNA duplex at the point of
transcription.
Transcription elongation
Transcription, like DNA replication, always
proceeds in the 5` to 3` direction. That is, a single
base is added de novo and then new RNA
nucleotides are added to the 3-OH free end, as in
DNA replication.
However, unlike DNA polymerase, prokaryotic RNA
polymerase does not seem to proofread as it
proceeds.
Most transcripts originate using (ATP) and, to a
lesser extent, (GTP) (purine nucleoside
triphosphates) at the +1 site. (UTP) and (CTP)
(pyrimidine nucleoside triphosphates) are
disfavoured at the initiation site.
Differences between replication and
transcription
(1) ribonucleotides are used in RNA synthesis
rather than deoxyribonucleotides;
(2) U replaces T as the complementary base pair
for A in RNA;
(3) a primer is not involved in RNA synthesis;
(4) only a very small portion of the genome is
transcribed or copied into RNA, whereas the entire
genome must be copied during DNA replication;
and
(5) there is no proofreading function during RNA
transcription.
RNA polymerase only goes one direction from a
promoter and only one strand of DNA is used as a
template at any one time.
To provide this template strand, the initiation of
transcription involves a short unwinding of the
DNA double helix. This is accomplished in a
two-step fashion.
First, RNA polymerase binds to the promoter to
form the closed complex, which is relatively weak.
Then, the double-stranded DNA goes through a
conformational change to form the much stronger
open complex through opening of the base pairs at
the -10 sequence, as shown next
Elongation is the function of the RNA polymerase
core enzyme. RNA polymerase moves along the
template, locally “unzipping” the DNA double helix.
This allows a transient base pairing between the
incoming nucleotide and newly-synthesized RNA
and the DNA template strand.
As it is made, the RNA transcript forms secondary
structure through intra-strand base pairing. The
average speed of transcription is about 40
nucleotides per second, much slower than DNA
polymerase.
Other protein factors may bind to polymerase and
alter the rate of transcription and some specific
sequences are transcribed more slowly than others
are. Eventually, RNA polymerase must come to the
end of the region to be transcribed.
A cistron is a region of DNA that encodes a single
polypeptide chain. In bacteria, mRNA is usually
generated from an operon as a polycistronic
transcript (one that contains the information to
produce a number of different proteins).
The polycistronic transcript is translated as it is
being transcribed. This transcript is not modified
and trimmed, and it does not contain introns
(regions within the coding sequence of a transcript
that are removed before translation occurs).
Several different proteins are produced during
translation of the polycistronic transcript, one from
each cistron.
Termination of transcription
The elongation reactions continue until the RNA
polymerase encounters a transcription termination
signal. One type of termination signal involves the
formation of a hairpin loop in the transcript,
preceding a number of U residues (rho
independent).
The second type of mechanism for termination
involves the binding of a protein, the rho factor,
which causes release of the RNA transcript from
the template (rho dependent).
The signal for both termination processes is the
sequence of bases in the newly synthesized RNA.
 Terminators
Both types of terminators sequenced so far have
one thing in common: they include a sequence and
its inverted form separated by another short
sequence, all together forming an inverted-repeat
sequence.
Inverted repeats can form a stem-loop structure by
pairing complementary bases within the
transcribed messenger RNA.
both rho-dependent and rho-independent
terminators have the stem-loop structure in RNA
just before the last base transcribed.
rho-independent
terminators
Rho-independent terminators have a characteristic
structure, which features (a) A strong G-C rich stem
and loop, (b) a sequence of 4–6 U residues in the
RNA, which are transcribed from a corresponding
stretch of As in the template.
Uracil-adenine base pairs have two hydrogen bonds
and are thus less stable thermodynamically than
guanine-cytosine base pairs.
Perhaps, the uracil-adenine base pairs
spontaneously denature, releasing the transcribed
RNA and the RNA polymerase,
rho-dependent terminators
Rho-dependent terminators do not have the uracil
sequence after the stem-loop structure.
Termination depends on the action of rho, which
appears to bind to the newly forming RNA. In an
ATP-dependent process, rho binds at a rho
utilisation site on the nascent RNA strand and runs
along the mRNA at a speed comparable to the
transcription process itself.
Possibly, when RNA polymerase pauses at the
stem-loop structure, rho catches up to the
polymerase and unwinds the DNA-RNA hybrid,
letting the DNA, RNA, and polymerase fall free. Rho
can do this because it has DNA-RNA helicase
(unwinding) properties.
TRANSCRIPTION OF EUKARYOTIC
GENES
Eukaryotic transcription is more complex than
prokaryotic transcription.
transcription, is primarily localized to the nucleus,
where it is separated from the cytoplasm (in which
translation occurs)
This allows for the temporal regulation of gene
expression through the sequestration of the RNA in
the nucleus, and allows for selective transport of
RNAs to the cytoplasm, where the ribosomes reside
Eukaryotes—unlike prokaryotes, which have only
one RNA polymerase—have three RNA
polymerases.
Pre-Initiation
Promoter chromatin is transformed from a static to
a dynamic state upon gene activation.
Nucleosomes are rapidly removed and reassembled
in the activated state. In vivo, nucleosomes are
removed from promoter DNA for transcription.
Promoter DNA is made transiently available for
interaction with the RNA polymerase II (Pol II)
transcription machinery.
Histone acetyl transferases HAT acetylates the
lysine on histones thereby making histones less
negative which reduces the electrostatic affinity of
histones to DNA
Initiation
All three eukaryotic RNA polymerases (I, II, and III)
recognize a seven-base sequence, TATAAAA,
located at about -25 on the promoter DNA. It is
similar to the -10 sequence in prokaryotes and is
called the TATA box (or Hogness box).
The TATA box is bound by 34 kDa TATA binding
protein (TBP), which in turn binds several other
proteins called TBP-associated factors (TAFs). This
complex of TBP and TAFs is referred to as TFIID.
Binding of TFIID to the TATA box sequence is
thought to represent the first step in the formation
of the transcription complex on the promoter.
i. RNA Polymerase II and TFIIF bind to the
TFIID-TFIIB complex forming a minimal
transcription initiation complex.
ii. TFIIE and TFIIH bind to produce the
complete transcription initiation complex
or preinitiation complex (PIC).
Once the PIC is formed, the rate of
transcription can be enhanced or repressed
by other transcription factors.
Enhancing TFs called activators must bind
to DNA sequence elements called
enhancers.
Among the large number of promoters that have
been sequenced, a few lack the TATA box, yet are
still transcribed.
Transcription initiation in these promoters appears
to be controlled by a CT-rich area, called the
initiator element (Inr), at +1 of the transcript (close
to the transcription start site), coupled with a
downstream promoter element (DPE) at about +28
to +34 of the transcript.
In TATA-less promoters, TFIID requires both these
elements to bind. The initiator element has a
consensus sequence of TCA(G or T)T(T or C), and
the downstream promoter element has the
consensus sequence of (A or G)G(A or T)CGTG.
Sequences farther upstream from the start site
determine how frequently the transcription event
occurs.
Typical of these DNA elements are the GC and
CAAT boxes, so named because of the DNA
sequences involved.
Together, then, the promoter and
promoter-proximal cis-active upstream elements
confer fidelity and frequency of initiation upon a
gene.
A third class of sequence elements can either
increase or decrease the rate of transcription
initiation of eukaryotic genes.
These elements are called either enhancers or
repressors (or silencers), depending on which
effect they have.
Hormone response elements (for steroids, T3,
retinoic acid, peptides, etc) act as—or in
conjunction with—enhancers or silencers
Tissue-specific expression of genes (eg, the
albumin gene in liver, the haemoglobin gene in
reticulocytes) is also mediated by specific DNA
sequences.
Elongation
Transcription produces heterogeneous nuclear
mRNAs hnRNA from the DNA template which can
not leave the nucleus until processed.
hnRNA processing involves addition of a 5`-cap
and a poly(A) tail and splicing to join exons and
remove introns. The product, mRNA, then can
migrate to the cytoplasm, where it will direct
protein synthesis.
Cap may protect initial end of forming RNA strand
from endonuclease activity.
In absence of the poly-A tail, the RNA is rapidly
degraded.
Transcription and capping of
mRNA
At the 5 end of polymerase II transcripts, 7-methyl
guanosine is added in the “wrong” direction, 5` -
5`.
This cap allows the ribosome to recognize the
beginning of a messenger RNA.
The processing of hnRNA molecules is a site for
regulation of gene expression.
There are three different types of methyl caps,
shown in blue: CAP0 refers to the methylated
guanosine (on the nitrogen at the seven position,
N7) added in the 5` to 5` linkage to the mRNA;
CAP1 refers to CAP0 with the addition of a methyl
to the 22` carbon of ribose on the nucleotide (N1)
at the 5` end of the chain; and CAP2 refers to
CAP1 with the addition of another 2` methyl group
to the next nucleotide (N2).
The methyl groups are donated by
S-adenosylmethionine (SAM).
Addition of a Poly(a) tail
Enzymes cleave the transcript (hnRNA) at a point
10–20 nucleotides beyond an AAUAAA sequence,
forming the 3` end.
After this cleavage, a poly(A) tail that can be over
200 nucleotides in length is added to the 3`-end.
there is no corresponding poly(dT) sequence in the
DNA template that corresponds to this tail; it is
added post - transcriptionally.
ATP serves as the precursor for the sequential
addition of the adenine nucleotides. They are
added one at a time, with poly(A) polymerase
catalysing each addition.
Removal of Introns
Eukaryotic pre-mRNA transcripts contain regions
known as exons and introns.
Exons only, appear in the mature mRNA; introns
are removed from the transcript and are not found
in the mature mRNA.
The consensus sequences at the intron/exon
boundaries of the pre-mRNA are AGGU (AGGT in
the DNA).
The sequences vary to some extent on the exon
side of the boundaries, but almost all introns begin
with a 5` GU and end with a 3` AG
Because every 5` GU and 3` AG combination does
not result in a functional splice site, clearly other
features within the exon or intron help to define
the appropriate splice sites.
At least two types of splicing occur, although they
are related:
◦ self-splicing (via ribozymes)
◦ protein-mediated splicing.
During self-splicing, the U-A bond at the left (5`)
side of the intron is transferred to GTP. The U that
is now unbound displaces the G at the right (3`)
side of the intron, reconnecting the RNA with a U-U
connection and releasing the intron
Since all bonds are reversible transfers
(transesterications) rather than new bonds, no
external energy source is required. Self-splicing
introns of this type are called group I introns.
Self-splicing has also been found in genes in the
mitochondria of yeast.
These introns are referred to as group II introns
because they use a different mechanism of splicing
that does not require an external nucleotide.
Instead, the rst bond is transferred within the
intron to an adenosine, forming a lariat structure
In order for the lariat to form, the ribose of the
adenosine must make three phosphodiester bonds
Protein-Mediated Splicing (the
Spliceosome)
Nuclear ribonucleoproteins (snurps U1 to U6) bind to
the intron, causing it to form a loop. The complex is
called a splicesosome. The U1 snurp binds near the
first exon/intron junction, and U2 binds within the
intron in a region containing an adenine nucleotide
residue.
Another group of snurps, U4, U5, and U6, binds to the
complex, and the loop is formed. The phosphate
attached to the G residue at the 5`-end of the intron
forms a 2`–5` linkage with the 2`-hydroxyl group of
the adenine nucleotide residue.
Cleavage occurs at the end of the first exon, between
the AG residues at the 3’ end of the exon and the GU
residues at the 5` end of the intron. The complex
continues to be held in place by the spliceosome. A
second cleavage occurs at the 3`-end of the intron
after the AG sequence.
The exons are joined together. The intron, shaped like
a lariat, is released and degraded to nucleotides.
The splicing machinery for the majority of introns
also includes numerous other polypeptides called
auxiliary and splicing factors; the entire splicing
process requires about 50 polypeptides.
A second, less common, intron, called the
U12-dependent intron, with different consensus
sequences, also exists. It is removed by a similar
splicing process involving different snRNPs (U11,
U12) as well as many components shared with the
major spliceosome.
RNA editing
The term RNA editing describes those molecular
processes in which the information content in an
RNA molecule is altered through a chemical change
in the base makeup.
To date, such changes have been observed in
tRNA, rRNA, mRNA and microRNA molecules of
eukaryotes but not prokaryotes. RNA editing
occurs in the cell nucleus and cytosol, as well as in
mitochondria and plastids
The diversity of RNA editing phenomena includes
nucleoside modifications such as cytidine (C) to
uridine (U) and adenosine (A) to inosine (I)
deaminations, as well as non-templated nucleotide
additions and insertions