an enzyme system converts the genetic

information in a segment of double-stranded

DNA into an RNA strand with a base sequence
complementary to one of the DNA strands
 Three major kinds of RNA are produced.

 Messenger RNAs (mRNAs) encode the amino acid

sequence of one or more polypeptides specified by a
gene or set of genes.

 Transfer RNAs (tRNAs) read the information

encoded in the mRNA and transfer the appropriate
amino acid to a growing polypeptide chain during
protein synthesis.

 Ribosomal RNAs (rRNAs) are constituents of

ribosomes, the intricate cellular machines
that synthesize proteins
Transcription resembles replication in its
 fundamental chemical mechanism,

 its polarity (direction of synthesis), and

 its use of a template.

 transcription has initiation, elongation, and

termination
Transcription differs from replication in that it
 does not require a primer and,

 generally, involves only limited segments of a

DNA molecule. Additionally, within
transcribed segments only one DNA strand
serves as a template.
 RNA Is Synthesized by RNA Polymerases
 DNA-dependent RNA polymerase requires, in
addition to a
 DNA template,
 all four ribonucleoside 5-triphosphates (ATP, GTP,
UTP, and CTP) as precursors of the nucleotide units
of RNA,
 as well as Mg2.
 RNA polymerase elongates an RNA strand by
adding ribonucleotide units to the 3-hydroxyl
end, building RNA in the 5´→ 3´ direction.

 The 3´-hydroxyl group acts as a nucleophile,

attacking the phosphate of the incoming
ribonucleoside triphosphate and releasing
pyrophosphate. The overall reaction is :

 (NMP)n + NTP → (NMP)n+1 + PPi

RNA Lengthened RNA
 To better understand, remember the reaction of
DNA synthesis
 Unlike DNA polymerase, RNA polymerase
does not require a primer to initiate synthesis.

 Initiation occurs when RNA polymerase binds

at specific DNA sequences called promoters.

 The 5´-triphosphate group of the first residue

in a nascent (newly formed) RNA molecule is
not cleaved to release PPi, but instead remains
intact throughout the transcription process.
 Elongation of a transcript by E. coli RNA
polymerase proceeds at a rate of 50 to 90
nucleotides/s.

The strand that serves as template for RNA
synthesis is called the template strand.
The DNA strand complementary to the template,
 the nontemplate strand, or coding strand, is
identical in base sequence to the RNA transcribed
from the gene
 The DNA-dependent RNA polymerase of E. coli is
a large, complex enzyme with five core subunits
(α2, β, β´, ω Mr 390,000) and a sixth subunit, one of
a group designated σ, with variants designated by
size (molecular weight).

 The σ subunit binds transiently to the core and

directs the enzyme to specific binding sites on the
DNA.
 These six subunits constitute the RNA polymerase
holoenzyme . The RNA polymerase holoenzyme of
E. coli thus exists in several forms, depending on
the type of σ subunit. The most common subunit is
σ70 (Mr 70,000),.
 RNA polymerases lack a separate proofreading
3´→ 5´ exonuclease active site (such as that of many
DNA polymerases), and the error rate for
transcription is higher than that for chromosomal
DNA replication—approximately one error for every
104 to 105 ribonucleotides incorporated into RNA.
 Because many copies of an RNA are generally
produced from a single gene and all RNAs are
eventually degraded and replaced, a mistake in
an RNA molecule is of less consequence to the
cell than a mistake in the permanent
information stored in DNA.
 Many RNA polymerases, including bacterial
RNA polymerase and the eukaryotic RNA
polymerase II do pause when a mispaired base
is added during transcription, and they can
remove mismatched nucleotides from the 3
end of a transcript by direct reversal of the
polymerase reaction.
 an RNA polymerase binds to specific
sequences in the DNA called promoters, which
direct the transcription of adjacent segments of
DNA (genes).
 By convention, the DNA base pairs that
correspond to the beginning of an RNA
molecule are given positive numbers,
and those preceding the RNA start site are
given negative numbers.

 The promoter region thus extends between

positions -70 and +30.
 Although the sequences are not identical for all
bacterial promoters in this class, certain
nucleotides that are particularly common at each
position form a consensus sequence
 The consensus sequence at the -10 region is
(5)TATAAT(3);
the consensus sequence at the -35 region is
(5)TTGACA(3).
 A third AT-rich recognition element,
called the UP (upstream promoter) element,
occurs between positions -40 and -60 in the
promoters of certain highly expressed genes.
The UP element is bound by the α subunit of
RNA polymerase.
 The pathway of transcription initiation
 E. coli has other classes of promoters, bound by
RNA polymerase holoenzymes with different
subunits.

 An example is the promoters of the heat-shock

genes.

 The products of this set of genes are made at

higher levels when the cell has received an insult,
such as a sudden increase in temperature.

 RNA polymerase binds to the promoters of these

genes only when σ70 is replaced with the σ32 (Mr
32,000) subunit, which is specific for the heat-shock
promoters
 Termination of RNA Synthesis
E. coli has at least two classes of termination signals

1. ρ-independent terminators have two
distinguishing features.
 The first is a region that produces an RNA transcript
with self-complementary sequences, permitting the
formation of a hairpin structure centered 15 to 20
nucleotides before the projected end of the RNA
strand.
 The second feature is a highly conserved string
of three A residues in the template strand that
are transcribed into U residues near the 3´ end
of the hairpin.

 When a polymerase arrives at a termination

site with this structure, it pauses formation of
the hairpin structure in the RNA disrupts
several AUU base pairs in the RNA-DNA
hybrid segment and may disrupt important
interactions between RNA and the RNA
polymerase, facilitating dissociation of the
transcript .
Model for ρ-independent termination of transcription
in E. coli.
2. The ρ-dependent terminators lack the sequence of
repeated A residues in the template strand but usually
include a CA-rich sequence called a rut (rho utilization)
element. The protein associates with the RNA at
specific binding sites and migrates in the 5´→3´
direction until it reaches the transcription complex that
is paused at a termination site. Here it contributes to
release of the RNA transcript.

The protein has an ATP-dependent RNA-DNA helicase

activity that promotes translocation of the protein
along the RNA, and ATP is hydrolyzed by protein
during the termination process.
The detailed mechanism by which the protein
promotes the release of the RNA transcript is not
known.
 Eukaryotic Cells Have Three Kinds of Nuclear
RNA Polymerases

 RNA polymerase I (Pol I) is responsible for the

synthesis of only one type of RNA, a transcript
called preribosomal RNA (or pre-rRNA), which
contains the precursor for the 18S, 5.8S, and 28S
rRNAs .
 The principal function of RNA polymerase II
(Pol II) is synthesis of mRNAs and some
specialized RNAs. This enzyme can recognize
thousands of promoters that vary greatly in
sequence.
 RNA polymerase III (Pol III) makes tRNAs, the
5SrRNA, and some other small specialized
RNAs.
 Eukaryotic Pol II is a huge enzyme with 12
subunits.
 The largest subunit (RBP1) exhibits a high
degree of homology to the β´ subunit of
bacterial RNA polymerase.
 Another subunit (RBP2) is structurally similar
to the bacterial β subunit
 two others (RBP3 and RBP11) show some
structural homology to the two bacterial α
subunits.
 The largest subunit of Pol II also has an unusual
feature, a long carboxyl-terminal tail consisting of
many repeats of a consensus heptad amino acid
sequence –YSPTSPS–.
 There are 27 repeats in the yeast enzyme
(18 exactly matching the consensus) and 52 (21
exact) in the mouse and human enzymes. This
carboxylterminal domain (CTD) is separated from
the main body of the enzyme by an unstructured
linker sequence.
 Common sequences in promoters recognized
by eukaryotic RNA polymerase II
Transcription in prokaryotes.mp4
 Diverse Functions of TFIIH In eukaryotes,
TFIIH participate in the formation of the closed
complex during assembly of a transcription complex,
but some of its subunits are also essential components
of the separate nucleotide-excision repair complex.

When Pol II transcription halts at the site of a
DNA lesion, TFIIH can interact with the lesion
and recruit the entire nucleotide-excision repair
complex.
 Genetic loss of certain TFIIH subunits can produce
human diseases. Some examples are xeroderma
pigmentosum and Cockayne’s syndrome,
which is characterized by arrested growth,
photosensitivity, and neurological disorders
 DNA-Dependent RNA Polymerase Undergoes
Selective Inhibition
 Actinomycin D The planar portion of this molecule
inserts (intercalates) into the double helical DNA
between successive G≡C base pairs,
deforming the DNA.
 This prevents movement of the polymerase along the
template. Because actinomycin D inhibits RNA
elongation in intact cells as well as in cell
extracts, it is used to identify cell processes that
depend on RNA synthesis.

Acridine inhibits RNA synthesis in a similar fashion

 Rifampicin inhibits bacterial RNA synthesis by
binding to the subunit of bacterial RNA
polymerases, preventing the promoter
clearance step of transcription. It is sometimes
used as an antibiotic.
 The mushroom Amanita phalloides has evolved
a very effective defense mechanism against
predators. It produces α-amanitin, which
disrupts mRNA formation in animal cells by
blocking Pol II and, at higher concentrations,
Pol III. Neither Pol I nor bacterial RNA
polymerase is sensitive to α-amanitin—nor is
the RNA polymerase II of A. phalloides itself!
 RNA Processing

A newly synthesized RNA molecule is called a primary
transcript
extensive processing of primary transcripts occurs in
eukaryotic mRNAs
and in tRNAs of both bacteria and eukaryotes

 The primary transcript for a eukaryotic mRNA typically

contains sequences encompassing one gene, although the
sequences encoding the polypeptide may not
be contiguous.
 Noncoding tracts that break up the coding region of the
transcript are called introns, and the

 coding segments are called exons


 splicing, the introns are removed from the
primary transcript and the exons are joined to
form a con tinuous sequence that specifies a
functional polypeptide.
 A modified residue called a 5´ cap is added at
the 5´ end.
 The 3´ end is cleaved, and 80 to 250 A residues
are added to create a poly(A) tail.
 Eukaryotic mRNAs Are Capped at the 5´ End

Most eukaryotic mRNAs have a 5´ cap, a residue of

7-methylguanosine linked to the 5´-terminal residue
of the mRNA through an unusual 5,5-triphosphate
linkage.

 The 5´ cap helps protect mRNA from ribonucleases.

The cap also binds to a specific cap binding
complex of proteins and participates in binding
of the mRNA to the ribosome to initiate translation.
 RNA Catalyzes the Splicing of Introns

 There are four classes of introns.

 The first two, the group I and group II introns, differ in the
details of their splicing mechanisms but share one
surprising characteristic: they are self-splicing—no protein
enzymes are involved.

 Group I introns are found in some nuclear, mitochondrial,

and chloroplast genes coding for rRNAs, mRNAs, and
tRNAs.

 Group II introns are generally found in the primary

transcripts of mitochondrial or chloroplast mRNAs in fungi,
algae, and plants. Group I and group II introns are also
found among the rarer examples of introns in bacteria
 Splicing mechanism of group I introns.
 Splicing mechanism of group II introns.
 The third and largest class of introns includes those
found in nuclear mRNA primary transcripts. These are
called spliceosomal introns,

 because their removal occurs within and is catalyzed by

a large protein complex called a spliceosome.

 The spliceosome is made up of specialized RNA-

protein complexes, small nuclear ribonucleoproteins .
Each snRNP contains one of a class of eukaryotic
RNAs, 100 to 200 nucleotides long, known as small
nuclear RNAs (snRNAs)
 The fourth class of introns, found in certain tRNAs, is
distinguished from the group I and II introns in that
the splicing reaction requires ATP and an
endonuclease.

 The splicing endonuclease cleaves the

phosphodiester bonds at both ends of the intron, and
the two exons are joined by a mechanism similar to
the DNA ligase reaction
 Eukaryotic mRNAs Have a Distinctive 3´ End
Structure

At their 3´ end, most eukaryotic mRNAs have a
string of 80 to 250 A residues, making up the
poly(A) tail.

This tail serves as a binding site for one or more
specific proteins. The poly(A) tail and its
associated proteins probably help protect mRNA
from enzymatic destruction.
 Addition of the poly(A) tail to the primary RNA
transcript of eukaryotes
 Overview of the processing of a eukaryotic
mRNA
 A Gene Can Give Rise to Multiple Products
by Differential RNA Processing
 (a) Alternative cleavage and polyadenylation patterns
b)Alternative splicing patterns two different 3´ splice sites
 Alternative processing of the calcitonin gene
transcript in rats.
 Processing of pre-rRNA transcripts in bacteria
 RNA-Dependent Synthesis of RNA and DNA

Retroviral infection of a mammalian cell and
integration of the retrovirus into the host
chromosome.

The RNA viruses that

contain reverse
transcriptases are
known as retroviruses
(retro is the Latin
prefix for “backward”)
 Reverse transcriptases catalyze three different
reactions:
(1) RNA-dependent DNA synthesis,
(2) RNA degradation, and
(3) DNA-dependent DNA synthesis
 Some Retroviruses Cause Cancer and AIDS

Most retroviruses do not kill their host cells but
remain integrated in the cellular DNA,
replicating when the cell divides. Some
retroviruses, classified as RNA tumor
viruses, contain an oncogene that can cause the
cell to grow abnormally .
 The first retrovirus of this type to be studied
was the Rous sarcoma virus (also called avian
sarcoma virus )
 The human immunodeficiency virus (HIV), which
causes acquired immune deficiency syndrome
(AIDS), is a retrovirus. Identified in 1983, HIV has
an RNA genome with standard retroviral genes
along with several other unusual genes (Fig. 26–32).

 The reverse transcriptase of HIV is even more error

prone than other known reverse transcriptases—ten
times more so— resulting in high mutation rates in
this virus. One or more errors are generally made
every time the viral genome is replicated, so any
 two viral RNA molecules are likely to differ!
 Because of the high error rate of the HIV
reverse transcriptase, the env gene in this virus
(along with the rest of the genome) undergoes
very rapid mutation, complicating the
development of an effective vaccine .
 Telomerase Is a Specialized Reverse Transcriptase

Telomeres, the structures at the ends of linear
eukaryotic chromosomes, generally consist of
many tandem copies of a short oligonucleotide
sequence.

 This sequence usually has the form TxGy in one

strand and CyAx in the complementary strand, where
x and y are typically in the range of 1 to 4 .

Telomeres vary in length from a few dozen base pairs
in some ciliated protozoans to tens of thousands of
base pairs in mammals. The TG strand is longer than
its complement, leaving a region of single-stranded
DNA of up to a few hundred nucleotides at the 3 end.
 Telomerase,, contains both RNA and protein
components.

 The RNA component is about 150 nucleotides long

and contains CyAx telomere repeat.
 This region of the RNA acts as a template for
synthesis of the TxGy strand of the telomere.

 Telomerase thereby acts as a cellular

reverse transcriptase that provides the active site for
RNA-dependent DNA synthesis
 After extension of the TxGy strand by
telomerase,
the complementary CyAx strand is synthesized
by cellular DNA polymerases, starting with an
RNA primer
 The single-stranded region is protected by specific
binding proteins in many lower eukaryotes,
especially those species with telomeres of less
than a few hundred base pairs.

 In higher eukaryotes (including mammals) with

telomeres many thousands of base pairs long, the
single-stranded end is sequestered in a specialized
structure called a T loop.

 The single stranded end is folded back and paired

with its complement in the double-stranded
portion of the telomere.

 The formation of a T loop involves invasion of the

3´ end .
 In mammals, the looped DNA is bound by two
proteins, TRF1 and TRF2, with the latter
protein involved in formation of the T loop.

 T loops protect the 3´ ends of chromosomes,

making them inaccessible to nucleases and the
enzymes that repair double-strand breaks
Telomerase Function - Animation.mp4
 In protozoans (such as Tetrahymena), loss of
telomerase activity results in a gradual
shortening of telomeres with each cell division,
ultimately leading to the death of the cell line.
 Some Viral RNAs Are Replicated by RNA-
Dependent RNA Polymarase
 Some E. coli bacteriophages, including f2, MS2,
R17, and Q, as well as some eukaryotic viruses
(including influenza and Sindbis viruses, the
latter associated with a form of encephalitis)
have RNA genomes.
 RNA Polymerase RNA-dependent RNA
polymerase (RNA replicase)

 The RNA replicase of most RNA bacteriophages

• molecular weight of ~210,000
• four subunits.
 One subunit (Mr 65,000) active site for replication.
 The other three subunits are host proteins normally
involved in host-cell protein synthesis:
 the E. coli elongation factors Tu (Mr 30,000) and Ts (Mr
45,000)(which ferry amino acyl–tRNAs to the
ribosomes) and
 protein S1 (an integral part of the 30S ribosomal subunit)