Download RNA SYNTHESIS

RNA SYNTHESIS, PROCESSING, AND MODIFICATION
MAJOR RNA CLASSES
FLOW OF GENETIC INFORMATION :
THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
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Ribosomal RNA make up 80% of the total RNA in the cell
Transfer RNA, the smallest of the 3 major RNA species
(excluding the small RNAs), make up 15% of the total
RNA
Messenger RNA comprises only 2-5%, but it is the most
heterogenous in terms of size and base sequence
Finally, the small RNAs are involved in mRNA splicing and
gene regulation
WHAT IS RNA?
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RNA is a polymer composed of alternating units of
ribonucleotides connected through a 3’-5’
phosphodiester bond.
In contrast with DNA, ribonucleotides contain:
o hydroxyl groups on the 2’-carbon of the ribose
sugar
o the base uracil in place of thymine
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SIMILARITIES BETWEEN DNA AND RNA
SYNTHESIS
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SIGNIFICANCE OF RNA
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The “working copies” of DNA
Expresses the master plan contained in DNA
Prokaryotic rRNA: 23S, 16S, 5S
Eukaryotic rRNA: 28S, 18S, 5.8S, 5S
“S” refers to a Svedberg Unit, which is a measure of size
based upon the molecular sedimentation rate during
ultracentrifugation
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Both have the general steps of initiation, elongation, and
termination with 5' to 3' polarity (synthesized in a 5’  3’
direction, antiparallel to the DNA template strand which
is read in a 3’  5’ direction)
Both have large, multicomponent initiation complexes
Both adhere to Watson-Crick base-pairing rules
Ribose used in RNA synthesis rather than deoxyribose
U replaces T as the complementary base pair for A in
RNA
A primer is not involved in RNA synthesis
Only a portion of the genome is transcribed or copied
into RNA, whereas the entire genome must be copied
during DNA replication
There is no proofreading function during RNA
transcription
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A transcription unit includes a promoter, an RNA-coding
region, and a terminator
The RNA product, which is synthesized in the 5' to 3'
direction, is the primary transcript
The template strand is always read in the 3' to 5'
direction
The opposite strand is called the coding strand
Within the DNA molecule, regions of both strands serve
as templates for specific RNA molecules
However, only one of the 2 DNA strands serves as a
template within a specific stretch of double helix
DNA DEPENDENT RNA POLYMERASE
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The enzyme responsible for the polymerization of
ribonucleotides into a sequence complementary to the
template strand of the gene
The enzyme attaches to the promoter on the template
strand
This is followed by initiation of RNA synthesis at the
starting point, and the process continues until a
termination sequence is reached
TRANSCRIPTION BY PROKARYOTIC RNA
POLYMERASE INVOLVES:
GENERAL FEATURES OF GENES
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The starting point of transcription corresponds to the 5'
nucleotide of the mRNA.
This is designated position +1, as is the corresponding
nucleotide in the DNA.
The numbers increase as the sequence proceeds
downstream
The nucleotide in the promoter adjacent to the
transcription initiation site is designated -1, and these
negative numbers increase as the sequence proceeds
upstream, away from the initiation site
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DEFINITIONS AND CONVENTIONS
A core enzyme, with its 4 subunits (plus an omega
subunit which is not shown), responsible for 5’  3’ RNA
polymerase activity
This enzyme lacks specificity (cannot recognize the
promoter on the DNA template)
The holoenzyme
o (s + core enzyme) contains the sigma subunit or
sigma factor (s) that enables RNA polymerase to
recognize promoter regions on the DNA
TERMINATION FACTOR
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A transcription unit is defined as that region of DNA that
includes the signals for transcription initiation,
elongation, and termination
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An auxiliary protein of RNA polymerase
Some regions of DNA that signal the termination of
transcription are recognized by RNA polymerase itself
Others are recognized by specific termination factors, an
example of which is the rho (r) factor of E. coli
E.COLI RNA POLYMERASE
- 35 SEQUENCE
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The primary transcripts generated by RNA polymerase II
(one of three nuclear DNA-dependent RNA polymerases
in eukaryotes) are promptly capped by 7methylguanosine triphosphate
These caps are necessary for the subsequent processing
of the primary transcript to mRNA, for the translation of
the mRNA, and for protection of the mRNA against
exonucleolytic attack
1) INITIATION
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A second consensus nucleotide sequence centered
around 35 bases to the left of the transcription start site
A mutation in either the Pribnow box or -35 sequence can
affect the transcription of the gene controlled by the mutant
promoter
2) ELONGATION
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Involves binding of the RNA polymerase holoenzyme to a
promoter region
Once the promoter has been recognized by the
holoenzyme, RNAP begins to synthesize a transcript of
the DNA sequence (usually beginning with a purine), and
the sigma subunit is released
Unlike DNA polymerase, RNA polymerase does not
require a primer and has no endo or exonuclease activity
(No repair capability)
GENERAL MECHANISM OF RNA SYNTHESIS
CONSENSUS NUCLEOTIDE SEQUENCES
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Pribnow box and -35 sequence
o Highly conserved
o Recognized by prokaryotic RNA polymerase sigma
factors
PRIBNOW BOX
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A stretch of 6 nucleotides (5’-TATAAT-3’) centered
around 8 to 10 nucleotides to the left of the transcription
start site that codes for the initial base of mRNA
elongation by addition of ribonucleotides to the 3’-OH
end
3’-OH acts as a nucleophile, attacking the -phosphate of
the incoming ribonucleoside triphosphate and releasing
pyrophosphate
mechanism is the same as that used for elongation of a
DNA strand
MECHANICAL FEATURES OF RNA SYNTHESIS
RHO-INDEPENDENT TERM INATION
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ROLE OF TOPOISOMERAS ES
3) TERMINATION
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Happens when a termination signal is reached
A rho factor protein may be required for the release of
the RNA product
Alternatively, the tetrameric RNA polymerase can
recognize termination regions on the DNA template
RHO DEPENDENT TERMIN ATION
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Requires rho factor protein
Rho factor binds to a C-rich region near the 3’ end of the
newly synthesized RNA, and migrates along behind the
RNA polymerase in the 5’ to 3’ direction until the
termination site is reached
Rho factor has ATP-dependent RNA-DNA helicase activity
that hydrolyzes ATP, and uses the energy to unwind the
3’ end of the transcript from the template
At the termination site, rho factor displaces the DNA
template strand, facilitating the dissociation of the RNA
molecule
Requires that the newly synthesized RNA have:
o A stable hairpin turn that slows down the progress
of RNA polymerase and causes it to pause
temporarily
o A hairpin turns complementary to a region of the
DNA template near the termination region that
exhibits 2-fold symmetery as a result of the
presence of a PALINDROME
EUKARYOTIC RNA POLYMERASES
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TRANSCRIPTION OF EUKARYOTIC GENES
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In addition to RNAP recognizing the promoter region and
initiating RNA synthesis, several supplemental
transcription factors (TFs) bind to DNA in eukaryotes
For RNAP and the TFs to recognize and bind to the
specific DNA sequence, the double helix must assume a
loose conformaton and dissociate temporarily from the
nucleosome core
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Unlike the E. coli RNA polymerase holoenzyme, each of
these require a number of additional proteins called
“transcription factors” in order to specifically bind to a
promoter and initiate transcription.
Eukaryotic promoters are composed of a variety of
different cis sequence elements which recruit some of
these trans-acting factors through DNA-protein
interactions.
Protein-protein interactions also occur and account for
many of the multi-component complexes found at
eukaryotic promoters.
Pol I and III promoters utilize a small number of
ubiquitous transcription factors while Pol II uses a large
variety of specific ones.
INITIATION AT RNA POL I PROMOTERS
CHROMATIN STRUCTURE AND GENE EXPRESSION
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Most actively transcribed genes are found in a relaxed
form of chromatin (EUCHROMATIN)
Most inactive segments are in a highly condensed
HETEROCHROMATIN
The interconversion of active and inactive forms of
chromatin is called CHROMATIN MODELING
Generally, genes that are inactive contain more
Methylated DNA (5-methylcytosine)
When histones become acetylated, the chromatin
structure becomes looser
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One model:
o Two identical subunits of the upstream binding
factor bind to the upstream core element and the
core promoter element.
o Protein:protein interactions between UBF
molecules force these two DNA sequences to come
into close proximity.
o This enables subsequent binding of selectivity factor
I, which consists of four subunits.
o Ultimately, this stabilized structure permits binding
of other factors (not shown), and finally RNA pol I.
INITIATION AT RNA POL III PROMOTERS
PROMOTERS FOR THE CLASS II GENES
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Positions of promoter elements in tRNA and 5S rRNA
genes.
Initiation of transcription of a tRNA gene:
The TFIIIC transcription factor binds via recognition of the
A and B sites
This permits subsequent binding of the trimeric TFIIIB
factor immediately upstream of the transcription start
site.
In response to TFIIIB binding, RNA polymerase III is
recruited and initiates transcription.
In the case of 5S rRNA genes, the process is similar,
except that an additional factor, TFIIIA, is required. TFIIIA
binds the C box, which permits subsequent binding of
TFIIIB and TFIIIC, then recruitment of RNA pol III.
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A sequence of DNA nucleotides that is almost identical to
the Pribnow box is usually found centered about 25
nucleotides upstream of the initial base of the
transcription start site for an mRNA molecule
This consensus sequence is the TATA or HOGNESS BOX
The consenus sequence of this element is TATAAAA (so it
resembles the TATAAT sequence of the prokaryotic 10 region).
The TATA box appears to be more important for selecting
the startpoint of transcription (i.e. positioning the
enzyme) than for defining the promoter.
TFIID, which binds to the TATA box promoter element,
is the only transcription factor capable of binding to
specific sequences of DNA. TFIID consists of TATA
binding protein (TBP) and 14 TBP-associated factors
(TAFs).
Between 70 and 80 nucleotides upstream from the
transcription start site is often found a second consensus
sequence called the CAAT Box.
The transcription factor CTF or NF1 binds to the CAAT
box.
The GC box is a common element in eukaryotic class II
promoters. Its consensus sequence is GGGCGG.
It may be present in one or more copies which can be
located between 40 and 100 bp upstream of the
startpoint of transcription.
The transcription factor Sp1 binds to the GC box.
ENHANCERS
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Special cis-acting DNA sequences that increase the rate
of initiation of transcription by RNAP II
Must be on the same chromosome as the gene whose
transcription they stimulate
They can be located upstream (to the 5’ side) or
downstream (to the 3’ side) of the transcription start site
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They can be close to or thousands of base pairs away
from the promoter
They can occur on either strand of the DNA
Enhancers contain DNA sequences called RESPONSE
ELEMENTS that bind specific transcription factors called
ACTIVATORS
By bending or looping the DNA, enhancer-binding factors
can interact with transcription factors bound to a
promoter and with RNAP II, stimulate transcription
Silencers are similar to enhancers in that they act over
long distances to reduce the level of gene expression
ACTION OF DRUGS
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Rifampin inhibits transcription initiation by binding to the
beta subunit of prokaryotic RNA polymerase, preventing
phosphodiester bond formation
Dactinomycin (Actinomycin D) is used for tumor therapy;
it binds to the DNA template and interferes with the
movement of RNA polymerase along the DNA
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TRANSCRIPTION INHIBI TORS
actinomycin D, acridine:
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FORMATION OF THE PREINITIATION COMPLEX
(PIC) FOR POL II ON A TATA PROMOTER
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intercalate between successive G=C base pairs in
duplex DNA
inhibit transcriptional elongation in pro- and
eukaryotes
1) TFIID recognizes and binds to the TATA box
2) TFIIA binds and stabilizes TFIID binding
3) The RNA polymerase II holoenzyme assembles - possibly
in a stepwise manner - to form a preinitiation complex
 The order of assembly of transcription factors may be
TFIID -> TFIIA -> TFIIB -> (TFIIF + RNAP II) -> TFIIE -> TFIIH
 Finally, the various regulatory factors (Srb-Mediator,
Srb10-CDK and Swi-Snf) bind to complete formation of
the pre-initiation complex.
rifampicin:
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POST-TRANSCRIPTIONAL RNA MODIFICATION
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Eukaryotic pol II differs from its prokaryotic counterpart
in that it has a series of heptad repeats with consensus
sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at the carboxyl
terminal of the largest pol II subunit.
This carboxyl terminal repeat domain (CTD) has 26
repeated units in brewers' yeast and 52 units in
mammalian cells.
The CTD is both a substrate for several kinases, including
the kinase component of TFIIH, and a binding site for a
wide array of proteins
Transcription factors are the ultimate targets of cellsignalling pathways. Whenever cells need to response to
an extracellular signal such as a hormone, the response is
mediated by a change in gene expression that comes
about, most often as the result of a change in the
phosphorylation state of a transcription factor.
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a-amanitin:
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Basal components
TBP, TFIIA, B, E, F, and H
Coactivators
TAFs (TBP + TAFs) = TFIID; Meds
Activators
SP1, ATF, CTF, AP1, etc
produced by fungus Amanita phalloides (death cap
mushroom)
potent inhibitor of RNA pol II and weak inhibitor of RNA
pol III
A primary transcript is the linear copy of the transcription
unit
The primary transcripts of both prokaryotic and
eukaryotic RNA post-transcriptionally modified by
cleavage using ribonucleases
tRNAs are further modified to help give each species a
unique identity
Prokaryotic mRNA is generally identical to its primary
transcript, while eukaryotic mRNA is extensively modified
after transcription
RIBOSOMAL RNA
CLASSES OF TRANSCRIP TION FACTORS IN
CLASS II GENES
General Mechanisms Specific Components
binds the b subunit of bacterial RNA polymerase
blocks promoter clearance (elongation)
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For prokaryotes & eukaryotes: synthesized from
preribosomal RNA
23S, 16S, and 5S ribosomal RNA of prokaryotes are
produced from a single RNA precursor molecule which is
processed in the nucleolus (same as Eukaryotic rRNA:
28S, 18S, 5.8S)
Eukaryotic 5S rRNA is synthesized by RNAP III and
modified separately
The preribosomal RNAs are cleaved by ribonucleases to
yield intermediate-sized pieces of rRNA, which are
further trimmed to produce the required rRNA species
TRANSFER RNA
POLY-A-TAIL
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tRNA is transcribed by RNA polymerase III
 The transcription product, the pre-tRNA, contains
additional RNA sequences at both the 5’ and 3’-ends
 These additional sequences are removed from the
transcript during processing
 The additional nucleotides at the 5’-end are
removed by an unusual RNA containing enzyme
called ribonuclease P (RNase P).
Some tRNA precursors contain an intron located in the
anticodon arm. These introns are spliced out during
processing of the tRNA.
CCA at the 3’-end
 These three bases are not coded for by the tRNA
gene. Instead, these nucleotides are added during
processing of the pre-tRNA transcript (by replacing
UU residues at the 3’ end)
 The enzyme responsible for the addition of the CCAend is tRNA nucleotidyl transferase
Base modifications are introduced into the tRNA at the
final processing step
 Addition of dihydrouridine, pseudouridine, and
methylated bases
 Modification of anticodon arm
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Most eukaryotic mRNAs have a chain of 40 to 200
adenine nucleotides attached to the 3’ end
Not transcribed from DNA but added by the nuclear
enzyme POLYADENYLATE POLYMERASE
A consensus sequence (AAUAAA) found near the 3’ end
signals that poly-A-tail must be added to stabilize RNA
and facilitate its exit from the nucleus
After entering the cytosol, the poly A tail is shortened
REMOVAL OF INTRONS
EUKARYOTIC MRNA
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The RNA molecule formed by RNAP II (the primary
transcript) contains the sequences found in cytosolic
mRNA
The collection of all the precursor molecules for mRNA is
known as HETEROGENOUS NUCLEAR RNA (hnRNA)
The 1ry transcripts are modified in the nucleus
5’ CAPPING
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7-methylguanosine is attached to the 5’ end of the mRNA
GTP addition is catalyzed by guanylyl transferase in the
nucleus
This is followed by methylation of the terminal guanine in
the cytosol, catalyzed by guanine-7-methyltransferase
SAM is the source of the methyl group
Cap addition permits the initiation of translation and
helps stabilize mRNA
Eukaryotic mRNA with no cap are not efficiently
translated
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Removal of RNA sequences that do not code for proteins
(intervening sequences) from the 1ry transcript
The remaining EXONS are spliced together by
SPLICEOSOME to form mature mRNA
The intron loops out as snRNPs (small nuclear
ribonucleoprotein particles or snurps, which are
complexes of snRNAs and proteins) bind to form the
spliceosome.
The intron is excised, and the exons are then spliced
together. The excised intron is released as a “lariat”
structure and degraded
The resulting mature mRNA may then exit the nucleus
and be translated in the cytoplasm.
Removal of introns from pre-mRNA transcripts involves
cleavage at the 5'- end of the intron by attack of a
specific 2'OH group, the branch site.
This forms a phosphodiester bond with the 5'-phosphate
of the intron, creating a lariat structure.
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The intron lariat is then removed, proceeding by attack of
3'-OH on Exon 1 to displace the intron from the 5'phosphate of Exon 2.
The GU and AG sequences at the branch site are
invariant
DISEASES
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The fatal disease Systemic Lupus Erythematosus (SLE)
results from an autoimmune response where the patient
produces antibodies against host proteins, including
snRNPs
Mutations that cause the incorrect splicing of beta-globin
mRNA are responsible for some cases of betathalassemia