Chapter 26 RNA Metabolism
1,How is RNA synthesized using DNA templates
(transcription)?
2,How is newly synthesized primary RNA
transcripts further processed to make
functional RNA molecules?
3,How is RNA and DNA synthesized using RNA
as template (reverse transcription);
4.What is the evolutionary implication of the
structural and functional complexity of RNA
molecules?
1,RNA molecules have great
structural and functional diversity
? With structures comparable to proteins in
complexity and uniqueness.
? Function as messengers between DNA and
polypeptides (mRNA),adapters (tRNA) to match a
specific amino acid with its specific genetic code
carried on mRNA,and the structural and catalytic
components of the protein-synthesizing ribosomes
(rRNA).
? Stores genetic information in RNA viruses.
? Catalyzes the processing of primary RNA transcripts.
? Might have appeared before DNA during evolution,
2,DNA and RNA syntheses are
similar in some aspects but different
in others
? Similar in fundamental chemical mechanism,both are
guided by a template; both have the same polarity in strand
extension (5` to 3`); both use triphosphate nucleotides
(dNTP or NTP).
? Different aspects,No primers are needed; only involves a
short segment of a large DNA molecule; uses only one of
the two complementary DNA strands as the template strand;
no proofreading; subject to great variation (when,where and
how efficient to start).
3,The multimeric RNA polymerase
in E.coli has multiple functions
? The holoenzyme consists of five types of subunits
(a2bb’?s)and its is used to synthesize all the RNA
molecules in E,coli.
? The multiple functions include:
– searches for initiation sites on the DNA molecule
and unwinds a short stretch of DNA (initiation);
– selects the correct NTP and catalyzes the
formation of phosphodiester bonds (elongation);
– detects termination signals for RNA synthesis
(termination).
The E,coli RNA polymerase holoenzyme consists of
six subunits,a2bb’? s.
Possible catalytic subunits
Promoter
specificity
Enzyme assembly,
promoter recognition,
activator binding
Role unknown
(not needed in vitro)
36.5 kDa
151 155 11 kDa
(32-90 kDa)
4,RNA synthesis occurs in a
“moving” transcription bubble on
the DNA template
? Only a short RNA-DNA hybrid (~8 bp in bacteria)
is present through the transcription process.
? At each moment,a region of about 17 bp on the E,
coli DNA is unwound in the transcription bubble.
? The RNA chain is extended at a rate of 50-90
nucleotides/second by the E,coli RNA polymerase.
? Unwinding ahead of and rewinding behind of the
transcription bubble produces positive and negative
supercoils respectively on the DNA (relieved by the
action of topoisomerases).
5,RNA polymerase recognizes
specific promoter sequences on
DNA to initiate transcription
? Promoter sequences are located adjacent to genes.
? Promoters can be identified using,protection assays”
(e.g.,footprinting techniques),
? Promoters,although all bind to the same polymerase,
have quite variable DNA sequences (surprisingly),but
with two consensus sequences centered at –10 and –
35 positions (the first residue of the RNA is given +1).
? Promoters having sequences more similar to the
consensus are more efficient,and vice versa (from
studies of mutations and activity comparison).
The footprinting
technique
The
footprint
- protein + protein
randomly
Footprinting,Purified RNA polymerase (or other DNA binding protein) is first
mixed with isolated and labeled DNA fragment that is believed to bind to the added protein
Before that DNA is cut with nonspecific DNase.
In the absence of DNA-binding proteinIn the presence of DNA-binding protein
An actual footprinting
result (RNA polymerase
binding to a lac promoter)
The
footprints
Alignment of different promoter sequences from
E.coli genes,the –10 (the Pribnow box) and
–35 consensus region were revealed.
Sequences of the
coding DNA strand
is conventionally
shown
Add TTTACC N12 TATAAT N7 A
Present only in certain highly expressed genes
Promoter of E.coli Add gene
6,The s subunits enable the E.coli
RNA polymerase to recognize
specific promoter sites
? The RNA polymerase without the s subunit (i.e.,the
a2bb’?) is unable to start transcription at a promoter.
? The s subunit decreases the affinity of RNA
polymerase for general (non-promoter) regions of
DNA by a factor of 104.
? E.coli contains multiple s factors for recognizing
different promoters,e.g.,s70 for standard promoters;
s32 for heat-shock promoters; s54 for nitrogen-
starvation promoters.
? Each type of s factor allows the cell to coordinately
express a set of genes.
Standard
Heat-shock
Nitrogen
starvation
s70 for standard promoters;
s32 for heat-shock promoters;
s54 for nitrogen-starvation promoters
E.coli contains multiple s factors for
recognizing different types of promoters:
7,RNA polymerase unwinds the
template DNA then initiate RNA
synthesis
? The enzyme slides to a promoter region and forms a
more tightly bound,closed complex”.
? Then the polymerase-promoter complex has to be
converted to an,open complex”,in which a 12-15
bp covering the region from the AT-rich –10 site to
+3 site is unwound.
? The essential transition from a,closed” to an,open”
complex sets the stage for RNA synthesis,after
which the core polymerase moves away from the
promoter.
random
8,E.coli RNA polymerase stops
synthesizing RNA at specific
terminator DNA sequences
? Two classes of transcription terminators have been
identified in bacteria,one depends on r protein,the
other is r-independent.
? At the r independent terminator,the transcribed
RNA is able to form a stem-loop (palindromic in
DNA sequence) structure followed by a stretch of
Us (oligoA in DNA).
? The r-dependent terminator needs the r protein,
which has an ATP-dependent RNA-DNA helicase
activity,for stopping RNA synthesis.
? The r-dependent terminator DNA exhibit no
obvious sequence similarities (probably the RNA
polymerase detects noncontiguous structural
features?).
? The r-dependent terminator is more often found in
phages (where it was originally discovered),but
rarely in E.coli.
? In contrast to what was originally expected,the
active signals for stopping RNA synthesis in both r-
independent and r-dependent transcription
terminators lie in the newly synthesized RNA rather
than in the DNA template.
r-independent
terminator,a model
Palindrome DNA
sequences
Oligo Us
Stem-loop
(hairpin) structure
Transcription
terminator of
E.coli Add gene
Model for an r-dependent
terminator
9,Transcription is a highly
regulated process
? Transcription is the first step in the complicated and
energy-expensive pathway leading to protein
synthesis,an ideal target for regulating gene
expression.
? The RNA polymerase binds to each promoter in
very different efficiency.
? Protein factors binding to DNA sequences close or
distant to the promoters can promote (activator) or
repress (repressor) the synthesis of certain RNA
molecules.
10,Three kinds of RNA polymerases
(I,II,and III) have been revealed for
making RNAs in the nuclears of
eukaryotic cells
? Each is responsible for the transcription of a certain
groups of genes,rRNA,mRNA or tRNA genes.
? The enzymes are often identified by examining their
sensitivity towards a-amanitin (from a toxic
mushroom),
Eukaryotic RNA
polymerases
T y p e L o c a l i z a t i o n T r a n s c r i p t s E f f e c t t o w a r d s
a - a m a n i t i n
I
N u c l e o l u s
18S,5.8S,
28S r R N A
I n s e n s i t i v e
II
N u c l e o p l a s m
m R N A
s n R NA
S t r o n g l y
s e n s i t i v e
III
N u c l e o p l a s m
t R N A,
5 S RNA
I n h i b it e d b y h ig h
c o n c e n t r a t io n s
11,RNA polymerase II (Pol II) binds
to promoters of thousands of
protein-coding genes
? Many Pol II promoters contain a TATAAA
sequence (called a TATA box) at -30 position and
an initiator sequence (Inr) at +1 position.
? The preinitiation complex (including Pol II) is
believed to assemble at the TATA box,with DNA
unwound at the
? Inr sequence.
? However,many Pol II promoters lack a TATA or
Inr or both sequences!
General features of promoters for protein-coding
genes in higher eukaryotes
GC
box
CAAT
box
12,Pol II is helped by an arrays of
protein factors (called transcription
factors) to form an active
transcription complex at a promoter
? First the TATA-binding protein (TBP) binds to the
TATA box,then TFIIB,TFIIF-Pol II,TFIIE,and
TFIIH will be added in order forming the closed
complex at the promoter.
? TFIIH then acts as a helicase to unwind the DNA
duplex at the Inr site,forming the open complex.
? A kinase activity of TFIIH will phosphoryate the C-
terminal domain (CTD) of Pol II,which will initiate
RNA synthesis and release the elongation complex.
? TFIIE and TFIIH will be released after the
elongation complex moves forward for a short
distance.
? Elongation factors will then join the elongation
complex and will suppress the pausing or arrest of
the Pol II-TFIIF complex,greatly enhancing the
efficiency of RNA synthesis.
? The termination of transcription of Pol II happens by
an unknown mechanism.
? This basal process of initiating RNA synthesis by
Pol II is elaborately regulated by many cell or tissue
specific protein factors that will binds to the
transcription factors,mostly act in a positive way.
? When Pol II transcription stalls at a site of DNA
lesion,TFIIH will binds at the lesion site and
appears to recruit the entire nucleotide-excision
repair complex,
TBP
DNA
A proposed
model
for Pol II-
catalyzed
mRNA
synthesis
13.The action of RNA polymerases
can be specifically inhibited
? Three-ring-containing,planar antibiotic molecules
like actinomycin D intercalates between two
successive GC base pairs in duplex DNA,
preventing RNA polymerases (all types) to move
along the template (thus the elongation of RNA
synthesis).
? Rifampicin (an antibiotic) binds to the b subunit of
bacterial RNA polymerases,preventing the
initiation of RNA synthesis.
? a-amanitin blocks eukaryotic mRNA synthesis by
binding to RNA polymerase II.
14,RNA molecules are often further
processed after being synthesized
on the DNA template
? The primary transcripts of eukaryotic mRNAs are
often capped at the 5` end,spliced in the middle
(introns removed and exons linked),
polyadenylated at the 3` end.
? The primary transcripts of both prokaryotic and
eukaryotic tRNAs are cleaved from both ends,
spliced in some cases,and modified for many of
the bases and sugars.
15,An eukaryotic mRNA precursor
acquire a 5` cap shortly after
transcription initiates
? A GMP component (from a GTP) is joined to the 5` end of
the mRNA in a novel 5`,5`-triphosphate linkage.
? The guanine base is then methylated at the N-7.
? The 2`-OH groups of the 1st and 2nd nucleotides adjacent to
the 7-methylguanine cap may also be methylated in certain
organisms.
? The methyl groups are transferred from S-
adenosylhomocysteine.
A 5` cap is added
to eukaryotic
mRNAs Before
transcription
ends
The 5` cap found
on the eukaryotic
mRNAs
16,Most eukaryotic mRNAs have a
poly(A) tail at the 3`end
? The tail consists of 80 to 250 adenylate residues.
? The mRNA precursors are extended beyond the site
where poly(A) tail is to be added.
? An AAUAAA sequence was found to be present in
all mRNAs and marks (together with other signals at
the 3`end) the site for cleavage and poly(A) tail
addition (11 to 30 nucleotides on the 3`end of the
AAUAAA sequence).
? The specific endonuclease and polyadenylate
polymerase,and other proteins probably exist as a
multiprotein complex to catalyze this event.
A poly(A) tail
is usually added
at the 3` end of an
mRNA molecule
via a processing
step.
17,EM studies of mRNA-DNA hybrids
revealed the discontinuity of eukaryotic
genes
? Each gene was found to be a continuous fragment of
DNA in the bacterial genome.
? But Berget and Sharp (1977) observed single-
stranded DNA loops when examining adenovirus
mRNA-DNA hybrids by electron microscopy.
? Such single-stranded DNA loops was widely
observed when examining such RNA-DNA hybrids.
? Intron sequences were proposed to be present on the
template DNA sequences,which are removed during
RNA processing,with exons linked together
precisely.
? Almost all genes in vertebrates contain introns (but
histone genes does not).
? Many genes in certain yeasts do not contain introns.
? Introns are also found in a few bacterial and
archaebacterial genes (but far less common than in
eukaryotic cells).
EM studies of mRNA-DNA hybrids for the
chicken ovalbumin gene (the R-looping
technique)
18,Four classes of introns have
been revealed having different
splicing mechanisms
? Group I introns are found in some nuclear,
mitochondrial and chloroplast genes encoding
rRNAs,mRNAs,and tRNAs.
? Group II introns are often found in genes encoding
mRNAs in mitochondrial and chloroplast DNA of
fungi,algae,and plants.
? Group III introns (the largest group) are found in
genes encoding eukaryotic nuclear mRNAs.
? Group IV introns are found in genes encoding the
tRNAs in the nuclear genomic DNA of eukaryotes
19,Group I introns are self-splicing
and use a guanine nucleoside or
nucleotide as the cofactor
? The intron present in the rRNA precursor of
Tetrahymena was found to be removed by itself
without using any proteins (Thomas Cech,1982).
? The intron is removed and the two exons precisely
linked via two nucleophilic transesterification
reactions (with two 3`-OH group act as the
nucleophiles).
Group I introns
are removed by
self-splicing via
two nucleophilic
transesterification
reactions.
The predicted secondary
structure of the self-splicing
rRNA intron of Tetrahymena
The internal guide sequence
5` splice site
3` splice site
20,Group II introns also undergo
self-splicing using forming a lariat-
like intermediate
? But the 2`-OH group of an adenylate residue within
in removing intron played the role of the 3`-OH
group of the guanine nucleoside or nucleotide in
group I intron self-splicing.
Group II introns are
removed via self-
splicing with an
adenylate residue
of the removing
intron acts as the
nucleophile,forming
an lariate-like
Intermediate.
21,Type III introns are found in the
nuclear mRNA primary transcripts
and have the largest numbers
? The splicing exon-intron junctions,determined by
comparing the sequences of the genomic DNA with
that of the cDNA prepared from the corresponding
mRNA,in mRNA precursors are specified by
sequences at the two ends of the introns,begin with
GU and end with AG.
? Type III introns are removed via a very similar way
as that of type II introns except being helped by
several highly conserved small nuclear
ribonucleoproteins (snRNPs),each containing a
class of U-rich small nuclear RNAs (snRNAs).
Type III introns,found on nuclear mRNA primary
transcripts,are removed via the spliceosomes
22,group IV introns are found in
tRNA precursors and are removed
by endonuclease and RNA ligase
? The splicing endonuclease first cleaves the
phosphodiester bonds at both ends of the intron.
? ATP is needed for the RNA ligase activity to join
the two exons.
? The joining reaction is similar to the DNA ligase-
catalyzed reaction.
? The mechanism of cleaving group IV introns is
different from that of group I,II,and III introns,all
including two transesterification reactions.
Group IV introns are
spliced via the
action of specific
endonuclease and
RNA ligase.
RNA ligase
23,Alternative proteins may be
produced from one single gene via
differential RNA processing
? The multiple transcripts produced from such a gene
may have more than one site for cleavage and
polyadenylation (as for immunoglobulin heavy
chains),alternative splicing (as for the myosin
heavy chains in fruit flies),or both (as for the
calcitonin gene in rats).
? In different cells or at different stages of
development,the transcript may be processed
differently to produce different gene products
(proteins).
Multiple mRNAs (thus polypeptide
chains) can be produced via
differential RNA processing.
24,The different rRNA molecules of
both prokaryotes and eukaryotes are
generated from single pre-rRNAs
? The 16S,23S and 5S rRNAs (together with certain
tRNAs) in bacteria are all generated from a single
30S pre-rRNA (about 6.5 kb,transcribed by RNA
polymerase I).
? There are seven pre-rRNA genes in the E.coli
genome (each encoding a different tRNA).
? The 18S,28S and 5.8S rRNAs in eukaryotes are
generated from a single 40S pre-rRNA (~14 kb).
? The 5S rRNA in eukaryotic cells is generated
separately (transcribed by RNA polymerase III).
All the rRNAs are derived
from a single precursor in
prokaryotic cells.
The 18S,5.8S,and 28S rRNAs in eukaryotic cells are
derived from one pre-rRNA molecule (the processing
needs small nucleolar RNA-containing proteins).
25,Primary tRNA transcripts
undergo a series of
posttranscriptional processing
? The extra sequences at the 5` and 3` ends are
removed by RNase P and RNase D respectively,
? The RNA in RNaseP is catalytic (Altman,1983)
? Type IV introns are occasionally present in pre-
tRNAs in eukaryotic cells.
? The CCA sequence is generated at the 3` end by the
action of tRNA nucleotidyltransferase (having three
active sites for the three ribonucleotides added).
? Some of the bases in tRNA molecules are modified
by methylation,deamination,reduction and others.
The processing of the primary tRNA
transcripts include removal of the 5`
and 3` ends,addition of the CCA sequence
at the 3` end,modification of many bases,
and splicing of introns (in eukaryotic cells).
Some typical modificied bases found
In mature tRNA molecules.
26,More RNA molecules (ribozymes)
were found to be catalytic
? Catalytic RNA molecules were also found in the
virusoid RNA (called hammerhead ribozymes).
? RNAs in the spliceosomes (the U-rich RNAs) and
ribosomes are also believed to be catalytic.
? A specific 3-D structure is required for ribozymes
to be catalytic.
? Ribozymes often orient their substrates via base
pairing.
? The excised intron (414 nucleotides) of the pre-
rRNA of Tetrahymena is further processed to a
RNA fragment of 395 nucleotides named as L-19
IVS;(intervening sequence lacking 19 nucleotides)
? A portion of the internal guide sequence remains at
the 5` end of L-19 IVS and the guanosine binding
site is still intact.
? Dr,Cech reasoned that L-19 IVS might act on
external substrates.
? L-19 IVS is able to catalyze the lengthening of some
oligonucleotides,like a (C)5 oligomer,at the
expense of others (being both a nuclease and
polymerase).
L-19 IVS
functions as
a real catalyst
in the test tube
Incubation time (minutes)
Labeled substrate
RNA
Nuclease
activity
RNA
polymerase
activity
The M1 RNA
in ribonuclease
P is catalytic
The intron in
the pre-rRNA of
Tetrahemena
is self-spliced
27,The cellular mRNAs are
degraded at different rates
? The level of a protein in a cell is determined to some
extent by the level of its mRNA,which depends on a
balance of the rates on its synthesis and degradation.
? The half of lives of different mRNA molecules vary
greatly,from seconds to many cell generations.
? 3` hairpin and poly(A) tails have been shown to
increase halve lives of mRNAs,but multiple,
sometimes overlapping AUUUA sequences have
been shown to decrease halve lives.
? The 5` 3` exoribonuclease is probably the major
degrading enzyme for mRNAs.
? The polynucleotide phosphorylase may be
another enzyme degrading mRNAs.
? Polynucleotide phosphorylase was used to
synthesize RNA for the first time in the test tube
(Severo Ochoa shared the Nobel Prize with
Arthur Kornberg in 1959 for this discovery).
?(NMP)n+1 + Pi (NMP)n + NDP
? This enzyme was used to synthesize RNA
polymers of different sequences and frequencies
of bases for the elucidation of the genetic codes.
Average half lives of mRNA
molecules
Bacteria 1.5 minutes
Vertebrates 3 hours
28,Reverse transcriptases catalyze
the production of DNA from RNA
? The existence of this enzyme in retroviruses ( RNA
viruses) was predicted by Howard Temin in 1962,
and proved by Temin and David Baltimore in 1970.
? This enzyme catalyzes three reactions,
– RNA-directed DNA synthesis using tRNAs as primers;
– Degradation of the RNA template;
– DNA-directed DNA synthesis;
? The enzyme has no 3` 5` proofreading exonuclease
activity,thus generating high rate of mutations.
? This enzyme is widely used to synthesize
complementary DNAs (cDNAs) from mRNAs.
Reverse transcriptases catalyzes the
sythesis of DNA from RNA template.
29,Telomerase catalyzes the
synthesis of the repeating telomere
sequences (TxGy) using an internal
RNA template
? Telomeres consist of a few to a large number of
tandem copies of a short oligonucleotide sequence that
are located at the two ends of the linear chromosomal
DNAs,having a 3` single strand extension (on the TG
strand).
? Telomerase acts to prevent the chromosomal ends
from becoming shortened after each replication (the
end part of the lagging strand can not be duplicated).
? Telomerase is actually a reverse transcriptase,but
uses a short segment of an internal RNA molecule
(~150 nucleotides) as the template to extend the end.
? The CyAx strand (the lagging strand) is believed to
be synthesized by a DNA polymerase using a RNA
primer.
? The ends of a linear chromosome is often protected
by binding to specific proteins,forming a T loop
structure in higher eukaryotes,where the single-
stranded DNA is sequestered.
? The length of the telomere seems to be inversely
related to the life span of cells and individuals
(shortens as one ages).
Problem posed
in the replication
of linear DNA:
the end of one
daughter strand
will be shortened
after each round
of replication.
The,inchworm”
(尺蠖） model for
telomerase action
The T loop observed at
one end of a mammalian
chromosome.
30,Some viral RNAs are replicated
by RNA-directed RNA polymerase
? The RNA genomes of some viruses (having bacteria,
animals or plants as their hosts) are replicated using
RNA-directed RNA polymerases (or RNA
replicases).
? The RNA replicase from bacteriophage-infected
E.coli cells consists of subunits encoded both by the
viruses and the host genome,
? They have features similar to that of DNA-directed
RNA polymerases,but are usually specific for the
RNA of the specific viruses.
? RNA replicases do not have proofreading activities.
31,Self-replicating RNA molecules
might be important for life to be
produced at the very beginning
? The realization of the structural and functional
complexity of RNA led a few scientists to propose
in 1960s that RNA might have serves as both
information carrier and catalyst at the early stage
of evolution.
? Synthesis of the peptide bonds of proteins seems to
be catalyzed by the rRNA component of ribosomes.
? A self-replicating mechanism for a RNA molecule
can be proposed based on the studies of the self-
splicing process of group I introns.
? SELEX (systematic evolution of ligands by
exponential enrichment) techniques have been used
to select RNA molecules (from a random RNA
library) that bind to various biomolecules (including
amino acids,organic dys,nucleotides,
cyanocobalamin and others).
A model to explain the
RNA-dependent synthesis
of an RNA polymer from
oligonucleotide precursors.
An ATP-binding
RNA was generated
and isolated using
SELEX.
Summary
? Transcription shares the basic chemical mechanisms
with replication except,no primers required,no
proofreading activity exist.
? Transcription begins at specific promoter sequences
(which can be identified using footprinting
technique) and ends at specific terminator sequences
(being r-independent or dependent in bacteria,and
not well understood in eukaryotes).
? One multimeric RNA polymerase catalyzes the
synthesis of all the RNA molecules in bacteria and
different RNA polymerases are used to synthesize
the different types of RNAs in eukaryotes,
? Primary RNA transcripts are further processed,capped,
tailed,spliced and sometimes edited for the mRNAs;
ends removed and modified,bases modified for the
tRNAs; cleaved and sometimes spliced for the
rRNAs.
? Catalytic RNAs were discovered when studying
RNA processing (splicing of group I and II introns,
removal of the 5`end of pre-tRNAs).
? RNAs can act as real enzymes (L-19 IVS,
hammerhead ribozymes).
? DNA can be synthesized by using RNA as templates
in a reaction catalyzed by reverse transcriptase.
? The telomeres of eukaryotic chromosomes are
synthesized by the action of telomerase,using an
RNA as template.
? RNA replicase catalyzes the synthesis of RNA from
RNA templates.
? RNA is very likely the first type of
biomacromolecules produced during biochemical
evolution.
1,How is RNA synthesized using DNA templates
(transcription)?
2,How is newly synthesized primary RNA
transcripts further processed to make
functional RNA molecules?
3,How is RNA and DNA synthesized using RNA
as template (reverse transcription);
4.What is the evolutionary implication of the
structural and functional complexity of RNA
molecules?
1,RNA molecules have great
structural and functional diversity
? With structures comparable to proteins in
complexity and uniqueness.
? Function as messengers between DNA and
polypeptides (mRNA),adapters (tRNA) to match a
specific amino acid with its specific genetic code
carried on mRNA,and the structural and catalytic
components of the protein-synthesizing ribosomes
(rRNA).
? Stores genetic information in RNA viruses.
? Catalyzes the processing of primary RNA transcripts.
? Might have appeared before DNA during evolution,
2,DNA and RNA syntheses are
similar in some aspects but different
in others
? Similar in fundamental chemical mechanism,both are
guided by a template; both have the same polarity in strand
extension (5` to 3`); both use triphosphate nucleotides
(dNTP or NTP).
? Different aspects,No primers are needed; only involves a
short segment of a large DNA molecule; uses only one of
the two complementary DNA strands as the template strand;
no proofreading; subject to great variation (when,where and
how efficient to start).
3,The multimeric RNA polymerase
in E.coli has multiple functions
? The holoenzyme consists of five types of subunits
(a2bb’?s)and its is used to synthesize all the RNA
molecules in E,coli.
? The multiple functions include:
– searches for initiation sites on the DNA molecule
and unwinds a short stretch of DNA (initiation);
– selects the correct NTP and catalyzes the
formation of phosphodiester bonds (elongation);
– detects termination signals for RNA synthesis
(termination).
The E,coli RNA polymerase holoenzyme consists of
six subunits,a2bb’? s.
Possible catalytic subunits
Promoter
specificity
Enzyme assembly,
promoter recognition,
activator binding
Role unknown
(not needed in vitro)
36.5 kDa
151 155 11 kDa
(32-90 kDa)
4,RNA synthesis occurs in a
“moving” transcription bubble on
the DNA template
? Only a short RNA-DNA hybrid (~8 bp in bacteria)
is present through the transcription process.
? At each moment,a region of about 17 bp on the E,
coli DNA is unwound in the transcription bubble.
? The RNA chain is extended at a rate of 50-90
nucleotides/second by the E,coli RNA polymerase.
? Unwinding ahead of and rewinding behind of the
transcription bubble produces positive and negative
supercoils respectively on the DNA (relieved by the
action of topoisomerases).
5,RNA polymerase recognizes
specific promoter sequences on
DNA to initiate transcription
? Promoter sequences are located adjacent to genes.
? Promoters can be identified using,protection assays”
(e.g.,footprinting techniques),
? Promoters,although all bind to the same polymerase,
have quite variable DNA sequences (surprisingly),but
with two consensus sequences centered at –10 and –
35 positions (the first residue of the RNA is given +1).
? Promoters having sequences more similar to the
consensus are more efficient,and vice versa (from
studies of mutations and activity comparison).
The footprinting
technique
The
footprint
- protein + protein
randomly
Footprinting,Purified RNA polymerase (or other DNA binding protein) is first
mixed with isolated and labeled DNA fragment that is believed to bind to the added protein
Before that DNA is cut with nonspecific DNase.
In the absence of DNA-binding proteinIn the presence of DNA-binding protein
An actual footprinting
result (RNA polymerase
binding to a lac promoter)
The
footprints
Alignment of different promoter sequences from
E.coli genes,the –10 (the Pribnow box) and
–35 consensus region were revealed.
Sequences of the
coding DNA strand
is conventionally
shown
Add TTTACC N12 TATAAT N7 A
Present only in certain highly expressed genes
Promoter of E.coli Add gene
6,The s subunits enable the E.coli
RNA polymerase to recognize
specific promoter sites
? The RNA polymerase without the s subunit (i.e.,the
a2bb’?) is unable to start transcription at a promoter.
? The s subunit decreases the affinity of RNA
polymerase for general (non-promoter) regions of
DNA by a factor of 104.
? E.coli contains multiple s factors for recognizing
different promoters,e.g.,s70 for standard promoters;
s32 for heat-shock promoters; s54 for nitrogen-
starvation promoters.
? Each type of s factor allows the cell to coordinately
express a set of genes.
Standard
Heat-shock
Nitrogen
starvation
s70 for standard promoters;
s32 for heat-shock promoters;
s54 for nitrogen-starvation promoters
E.coli contains multiple s factors for
recognizing different types of promoters:
7,RNA polymerase unwinds the
template DNA then initiate RNA
synthesis
? The enzyme slides to a promoter region and forms a
more tightly bound,closed complex”.
? Then the polymerase-promoter complex has to be
converted to an,open complex”,in which a 12-15
bp covering the region from the AT-rich –10 site to
+3 site is unwound.
? The essential transition from a,closed” to an,open”
complex sets the stage for RNA synthesis,after
which the core polymerase moves away from the
promoter.
random
8,E.coli RNA polymerase stops
synthesizing RNA at specific
terminator DNA sequences
? Two classes of transcription terminators have been
identified in bacteria,one depends on r protein,the
other is r-independent.
? At the r independent terminator,the transcribed
RNA is able to form a stem-loop (palindromic in
DNA sequence) structure followed by a stretch of
Us (oligoA in DNA).
? The r-dependent terminator needs the r protein,
which has an ATP-dependent RNA-DNA helicase
activity,for stopping RNA synthesis.
? The r-dependent terminator DNA exhibit no
obvious sequence similarities (probably the RNA
polymerase detects noncontiguous structural
features?).
? The r-dependent terminator is more often found in
phages (where it was originally discovered),but
rarely in E.coli.
? In contrast to what was originally expected,the
active signals for stopping RNA synthesis in both r-
independent and r-dependent transcription
terminators lie in the newly synthesized RNA rather
than in the DNA template.
r-independent
terminator,a model
Palindrome DNA
sequences
Oligo Us
Stem-loop
(hairpin) structure
Transcription
terminator of
E.coli Add gene
Model for an r-dependent
terminator
9,Transcription is a highly
regulated process
? Transcription is the first step in the complicated and
energy-expensive pathway leading to protein
synthesis,an ideal target for regulating gene
expression.
? The RNA polymerase binds to each promoter in
very different efficiency.
? Protein factors binding to DNA sequences close or
distant to the promoters can promote (activator) or
repress (repressor) the synthesis of certain RNA
molecules.
10,Three kinds of RNA polymerases
(I,II,and III) have been revealed for
making RNAs in the nuclears of
eukaryotic cells
? Each is responsible for the transcription of a certain
groups of genes,rRNA,mRNA or tRNA genes.
? The enzymes are often identified by examining their
sensitivity towards a-amanitin (from a toxic
mushroom),
Eukaryotic RNA
polymerases
T y p e L o c a l i z a t i o n T r a n s c r i p t s E f f e c t t o w a r d s
a - a m a n i t i n
I
N u c l e o l u s
18S,5.8S,
28S r R N A
I n s e n s i t i v e
II
N u c l e o p l a s m
m R N A
s n R NA
S t r o n g l y
s e n s i t i v e
III
N u c l e o p l a s m
t R N A,
5 S RNA
I n h i b it e d b y h ig h
c o n c e n t r a t io n s
11,RNA polymerase II (Pol II) binds
to promoters of thousands of
protein-coding genes
? Many Pol II promoters contain a TATAAA
sequence (called a TATA box) at -30 position and
an initiator sequence (Inr) at +1 position.
? The preinitiation complex (including Pol II) is
believed to assemble at the TATA box,with DNA
unwound at the
? Inr sequence.
? However,many Pol II promoters lack a TATA or
Inr or both sequences!
General features of promoters for protein-coding
genes in higher eukaryotes
GC
box
CAAT
box
12,Pol II is helped by an arrays of
protein factors (called transcription
factors) to form an active
transcription complex at a promoter
? First the TATA-binding protein (TBP) binds to the
TATA box,then TFIIB,TFIIF-Pol II,TFIIE,and
TFIIH will be added in order forming the closed
complex at the promoter.
? TFIIH then acts as a helicase to unwind the DNA
duplex at the Inr site,forming the open complex.
? A kinase activity of TFIIH will phosphoryate the C-
terminal domain (CTD) of Pol II,which will initiate
RNA synthesis and release the elongation complex.
? TFIIE and TFIIH will be released after the
elongation complex moves forward for a short
distance.
? Elongation factors will then join the elongation
complex and will suppress the pausing or arrest of
the Pol II-TFIIF complex,greatly enhancing the
efficiency of RNA synthesis.
? The termination of transcription of Pol II happens by
an unknown mechanism.
? This basal process of initiating RNA synthesis by
Pol II is elaborately regulated by many cell or tissue
specific protein factors that will binds to the
transcription factors,mostly act in a positive way.
? When Pol II transcription stalls at a site of DNA
lesion,TFIIH will binds at the lesion site and
appears to recruit the entire nucleotide-excision
repair complex,
TBP
DNA
A proposed
model
for Pol II-
catalyzed
mRNA
synthesis
13.The action of RNA polymerases
can be specifically inhibited
? Three-ring-containing,planar antibiotic molecules
like actinomycin D intercalates between two
successive GC base pairs in duplex DNA,
preventing RNA polymerases (all types) to move
along the template (thus the elongation of RNA
synthesis).
? Rifampicin (an antibiotic) binds to the b subunit of
bacterial RNA polymerases,preventing the
initiation of RNA synthesis.
? a-amanitin blocks eukaryotic mRNA synthesis by
binding to RNA polymerase II.
14,RNA molecules are often further
processed after being synthesized
on the DNA template
? The primary transcripts of eukaryotic mRNAs are
often capped at the 5` end,spliced in the middle
(introns removed and exons linked),
polyadenylated at the 3` end.
? The primary transcripts of both prokaryotic and
eukaryotic tRNAs are cleaved from both ends,
spliced in some cases,and modified for many of
the bases and sugars.
15,An eukaryotic mRNA precursor
acquire a 5` cap shortly after
transcription initiates
? A GMP component (from a GTP) is joined to the 5` end of
the mRNA in a novel 5`,5`-triphosphate linkage.
? The guanine base is then methylated at the N-7.
? The 2`-OH groups of the 1st and 2nd nucleotides adjacent to
the 7-methylguanine cap may also be methylated in certain
organisms.
? The methyl groups are transferred from S-
adenosylhomocysteine.
A 5` cap is added
to eukaryotic
mRNAs Before
transcription
ends
The 5` cap found
on the eukaryotic
mRNAs
16,Most eukaryotic mRNAs have a
poly(A) tail at the 3`end
? The tail consists of 80 to 250 adenylate residues.
? The mRNA precursors are extended beyond the site
where poly(A) tail is to be added.
? An AAUAAA sequence was found to be present in
all mRNAs and marks (together with other signals at
the 3`end) the site for cleavage and poly(A) tail
addition (11 to 30 nucleotides on the 3`end of the
AAUAAA sequence).
? The specific endonuclease and polyadenylate
polymerase,and other proteins probably exist as a
multiprotein complex to catalyze this event.
A poly(A) tail
is usually added
at the 3` end of an
mRNA molecule
via a processing
step.
17,EM studies of mRNA-DNA hybrids
revealed the discontinuity of eukaryotic
genes
? Each gene was found to be a continuous fragment of
DNA in the bacterial genome.
? But Berget and Sharp (1977) observed single-
stranded DNA loops when examining adenovirus
mRNA-DNA hybrids by electron microscopy.
? Such single-stranded DNA loops was widely
observed when examining such RNA-DNA hybrids.
? Intron sequences were proposed to be present on the
template DNA sequences,which are removed during
RNA processing,with exons linked together
precisely.
? Almost all genes in vertebrates contain introns (but
histone genes does not).
? Many genes in certain yeasts do not contain introns.
? Introns are also found in a few bacterial and
archaebacterial genes (but far less common than in
eukaryotic cells).
EM studies of mRNA-DNA hybrids for the
chicken ovalbumin gene (the R-looping
technique)
18,Four classes of introns have
been revealed having different
splicing mechanisms
? Group I introns are found in some nuclear,
mitochondrial and chloroplast genes encoding
rRNAs,mRNAs,and tRNAs.
? Group II introns are often found in genes encoding
mRNAs in mitochondrial and chloroplast DNA of
fungi,algae,and plants.
? Group III introns (the largest group) are found in
genes encoding eukaryotic nuclear mRNAs.
? Group IV introns are found in genes encoding the
tRNAs in the nuclear genomic DNA of eukaryotes
19,Group I introns are self-splicing
and use a guanine nucleoside or
nucleotide as the cofactor
? The intron present in the rRNA precursor of
Tetrahymena was found to be removed by itself
without using any proteins (Thomas Cech,1982).
? The intron is removed and the two exons precisely
linked via two nucleophilic transesterification
reactions (with two 3`-OH group act as the
nucleophiles).
Group I introns
are removed by
self-splicing via
two nucleophilic
transesterification
reactions.
The predicted secondary
structure of the self-splicing
rRNA intron of Tetrahymena
The internal guide sequence
5` splice site
3` splice site
20,Group II introns also undergo
self-splicing using forming a lariat-
like intermediate
? But the 2`-OH group of an adenylate residue within
in removing intron played the role of the 3`-OH
group of the guanine nucleoside or nucleotide in
group I intron self-splicing.
Group II introns are
removed via self-
splicing with an
adenylate residue
of the removing
intron acts as the
nucleophile,forming
an lariate-like
Intermediate.
21,Type III introns are found in the
nuclear mRNA primary transcripts
and have the largest numbers
? The splicing exon-intron junctions,determined by
comparing the sequences of the genomic DNA with
that of the cDNA prepared from the corresponding
mRNA,in mRNA precursors are specified by
sequences at the two ends of the introns,begin with
GU and end with AG.
? Type III introns are removed via a very similar way
as that of type II introns except being helped by
several highly conserved small nuclear
ribonucleoproteins (snRNPs),each containing a
class of U-rich small nuclear RNAs (snRNAs).
Type III introns,found on nuclear mRNA primary
transcripts,are removed via the spliceosomes
22,group IV introns are found in
tRNA precursors and are removed
by endonuclease and RNA ligase
? The splicing endonuclease first cleaves the
phosphodiester bonds at both ends of the intron.
? ATP is needed for the RNA ligase activity to join
the two exons.
? The joining reaction is similar to the DNA ligase-
catalyzed reaction.
? The mechanism of cleaving group IV introns is
different from that of group I,II,and III introns,all
including two transesterification reactions.
Group IV introns are
spliced via the
action of specific
endonuclease and
RNA ligase.
RNA ligase
23,Alternative proteins may be
produced from one single gene via
differential RNA processing
? The multiple transcripts produced from such a gene
may have more than one site for cleavage and
polyadenylation (as for immunoglobulin heavy
chains),alternative splicing (as for the myosin
heavy chains in fruit flies),or both (as for the
calcitonin gene in rats).
? In different cells or at different stages of
development,the transcript may be processed
differently to produce different gene products
(proteins).
Multiple mRNAs (thus polypeptide
chains) can be produced via
differential RNA processing.
24,The different rRNA molecules of
both prokaryotes and eukaryotes are
generated from single pre-rRNAs
? The 16S,23S and 5S rRNAs (together with certain
tRNAs) in bacteria are all generated from a single
30S pre-rRNA (about 6.5 kb,transcribed by RNA
polymerase I).
? There are seven pre-rRNA genes in the E.coli
genome (each encoding a different tRNA).
? The 18S,28S and 5.8S rRNAs in eukaryotes are
generated from a single 40S pre-rRNA (~14 kb).
? The 5S rRNA in eukaryotic cells is generated
separately (transcribed by RNA polymerase III).
All the rRNAs are derived
from a single precursor in
prokaryotic cells.
The 18S,5.8S,and 28S rRNAs in eukaryotic cells are
derived from one pre-rRNA molecule (the processing
needs small nucleolar RNA-containing proteins).
25,Primary tRNA transcripts
undergo a series of
posttranscriptional processing
? The extra sequences at the 5` and 3` ends are
removed by RNase P and RNase D respectively,
? The RNA in RNaseP is catalytic (Altman,1983)
? Type IV introns are occasionally present in pre-
tRNAs in eukaryotic cells.
? The CCA sequence is generated at the 3` end by the
action of tRNA nucleotidyltransferase (having three
active sites for the three ribonucleotides added).
? Some of the bases in tRNA molecules are modified
by methylation,deamination,reduction and others.
The processing of the primary tRNA
transcripts include removal of the 5`
and 3` ends,addition of the CCA sequence
at the 3` end,modification of many bases,
and splicing of introns (in eukaryotic cells).
Some typical modificied bases found
In mature tRNA molecules.
26,More RNA molecules (ribozymes)
were found to be catalytic
? Catalytic RNA molecules were also found in the
virusoid RNA (called hammerhead ribozymes).
? RNAs in the spliceosomes (the U-rich RNAs) and
ribosomes are also believed to be catalytic.
? A specific 3-D structure is required for ribozymes
to be catalytic.
? Ribozymes often orient their substrates via base
pairing.
? The excised intron (414 nucleotides) of the pre-
rRNA of Tetrahymena is further processed to a
RNA fragment of 395 nucleotides named as L-19
IVS;(intervening sequence lacking 19 nucleotides)
? A portion of the internal guide sequence remains at
the 5` end of L-19 IVS and the guanosine binding
site is still intact.
? Dr,Cech reasoned that L-19 IVS might act on
external substrates.
? L-19 IVS is able to catalyze the lengthening of some
oligonucleotides,like a (C)5 oligomer,at the
expense of others (being both a nuclease and
polymerase).
L-19 IVS
functions as
a real catalyst
in the test tube
Incubation time (minutes)
Labeled substrate
RNA
Nuclease
activity
RNA
polymerase
activity
The M1 RNA
in ribonuclease
P is catalytic
The intron in
the pre-rRNA of
Tetrahemena
is self-spliced
27,The cellular mRNAs are
degraded at different rates
? The level of a protein in a cell is determined to some
extent by the level of its mRNA,which depends on a
balance of the rates on its synthesis and degradation.
? The half of lives of different mRNA molecules vary
greatly,from seconds to many cell generations.
? 3` hairpin and poly(A) tails have been shown to
increase halve lives of mRNAs,but multiple,
sometimes overlapping AUUUA sequences have
been shown to decrease halve lives.
? The 5` 3` exoribonuclease is probably the major
degrading enzyme for mRNAs.
? The polynucleotide phosphorylase may be
another enzyme degrading mRNAs.
? Polynucleotide phosphorylase was used to
synthesize RNA for the first time in the test tube
(Severo Ochoa shared the Nobel Prize with
Arthur Kornberg in 1959 for this discovery).
?(NMP)n+1 + Pi (NMP)n + NDP
? This enzyme was used to synthesize RNA
polymers of different sequences and frequencies
of bases for the elucidation of the genetic codes.
Average half lives of mRNA
molecules
Bacteria 1.5 minutes
Vertebrates 3 hours
28,Reverse transcriptases catalyze
the production of DNA from RNA
? The existence of this enzyme in retroviruses ( RNA
viruses) was predicted by Howard Temin in 1962,
and proved by Temin and David Baltimore in 1970.
? This enzyme catalyzes three reactions,
– RNA-directed DNA synthesis using tRNAs as primers;
– Degradation of the RNA template;
– DNA-directed DNA synthesis;
? The enzyme has no 3` 5` proofreading exonuclease
activity,thus generating high rate of mutations.
? This enzyme is widely used to synthesize
complementary DNAs (cDNAs) from mRNAs.
Reverse transcriptases catalyzes the
sythesis of DNA from RNA template.
29,Telomerase catalyzes the
synthesis of the repeating telomere
sequences (TxGy) using an internal
RNA template
? Telomeres consist of a few to a large number of
tandem copies of a short oligonucleotide sequence that
are located at the two ends of the linear chromosomal
DNAs,having a 3` single strand extension (on the TG
strand).
? Telomerase acts to prevent the chromosomal ends
from becoming shortened after each replication (the
end part of the lagging strand can not be duplicated).
? Telomerase is actually a reverse transcriptase,but
uses a short segment of an internal RNA molecule
(~150 nucleotides) as the template to extend the end.
? The CyAx strand (the lagging strand) is believed to
be synthesized by a DNA polymerase using a RNA
primer.
? The ends of a linear chromosome is often protected
by binding to specific proteins,forming a T loop
structure in higher eukaryotes,where the single-
stranded DNA is sequestered.
? The length of the telomere seems to be inversely
related to the life span of cells and individuals
(shortens as one ages).
Problem posed
in the replication
of linear DNA:
the end of one
daughter strand
will be shortened
after each round
of replication.
The,inchworm”
(尺蠖） model for
telomerase action
The T loop observed at
one end of a mammalian
chromosome.
30,Some viral RNAs are replicated
by RNA-directed RNA polymerase
? The RNA genomes of some viruses (having bacteria,
animals or plants as their hosts) are replicated using
RNA-directed RNA polymerases (or RNA
replicases).
? The RNA replicase from bacteriophage-infected
E.coli cells consists of subunits encoded both by the
viruses and the host genome,
? They have features similar to that of DNA-directed
RNA polymerases,but are usually specific for the
RNA of the specific viruses.
? RNA replicases do not have proofreading activities.
31,Self-replicating RNA molecules
might be important for life to be
produced at the very beginning
? The realization of the structural and functional
complexity of RNA led a few scientists to propose
in 1960s that RNA might have serves as both
information carrier and catalyst at the early stage
of evolution.
? Synthesis of the peptide bonds of proteins seems to
be catalyzed by the rRNA component of ribosomes.
? A self-replicating mechanism for a RNA molecule
can be proposed based on the studies of the self-
splicing process of group I introns.
? SELEX (systematic evolution of ligands by
exponential enrichment) techniques have been used
to select RNA molecules (from a random RNA
library) that bind to various biomolecules (including
amino acids,organic dys,nucleotides,
cyanocobalamin and others).
A model to explain the
RNA-dependent synthesis
of an RNA polymer from
oligonucleotide precursors.
An ATP-binding
RNA was generated
and isolated using
SELEX.
Summary
? Transcription shares the basic chemical mechanisms
with replication except,no primers required,no
proofreading activity exist.
? Transcription begins at specific promoter sequences
(which can be identified using footprinting
technique) and ends at specific terminator sequences
(being r-independent or dependent in bacteria,and
not well understood in eukaryotes).
? One multimeric RNA polymerase catalyzes the
synthesis of all the RNA molecules in bacteria and
different RNA polymerases are used to synthesize
the different types of RNAs in eukaryotes,
? Primary RNA transcripts are further processed,capped,
tailed,spliced and sometimes edited for the mRNAs;
ends removed and modified,bases modified for the
tRNAs; cleaved and sometimes spliced for the
rRNAs.
? Catalytic RNAs were discovered when studying
RNA processing (splicing of group I and II introns,
removal of the 5`end of pre-tRNAs).
? RNAs can act as real enzymes (L-19 IVS,
hammerhead ribozymes).
? DNA can be synthesized by using RNA as templates
in a reaction catalyzed by reverse transcriptase.
? The telomeres of eukaryotic chromosomes are
synthesized by the action of telomerase,using an
RNA as template.
? RNA replicase catalyzes the synthesis of RNA from
RNA templates.
? RNA is very likely the first type of
biomacromolecules produced during biochemical
evolution.
