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RNA and RNA world
WHICH CAME FIRST,
THE chicken or the egg?
The biological silences
have a variation: which
came first, DNA or
protein? You see,
among the many tasks
performed by proteins
is assembling DNA
molecules. But DNA
contains the in
formation needed to
make proteins. So
which came first?
Transcription
• Transcription-the synthesis of RNA using
DNA as a template.
• Four stages: Initiation, Elongation,
Termination, Post-transcriptional
modification
Similarities and Differences from
Replication
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•
•
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Similarities
Differences
5' →3' direction
•RNA polymerase instead
many proteins involved of DNA polymerase
initiation, elongation, • no proofreading
termination
• posttranscriptional
• transcription bubble
modification
• starts and stops at
• 1 strand copied not 2
specific places
• not all copied
Nt not dNt
Terminology of Transcription
(3') ACG GAA TAA TTA TGT TTA TCA CAA TTT AGA (5') + (plus) strand
template strand
(5') TGC CTT ATT AAT ACA AAT AGT GTT AAA TCT (3') -(minus) strand
non-template strand
coding strand
(5') UGC CUU AUU AAU ACA AAU AGU GUU AAA UCU (3') RNA
transcript
Upstream 5’ end vs. Downstream 3’ end
• The function of RNA polymerase is to copy one strand of
duplex DNA into RNA.
Transcription in prokaryotes
Transcription Unit
Optimal Promoter
T82 T84 G78 A65 C54 A45
T80 A95 T45 A60 A50 T96
The –35 sequence is used for initial recognition, and the –10 sequence
is used for the melting reaction that converts a closed complex to an
open complex.
• During transcription, the bubble is maintained within bacterial RNA
polymerase, which unwinds and rewinds DNA, maintains the conditions
of the partner and template DNA strands, and synthesizes RNA.
Bacterial RNA Polymerases
How does RNA polymerase work?
A single type of RNA polymerase is
responsible for almost all synthesis of
mRNA, rRNA and tRNA in a eubacterium.
About 7,000 RNA polymerase molecules
are present in an E. coli cell. Probably
2,000~5,000 enzymes are synthesizing RNA
at any one time, the number depending on
the growth conditions.
How many sigma
factors exist
in E. coli ?
A map of the E. coli s70 factor
identifies conserved regions
How does transcription initiate?
Four stages of Transcription
Termination
• Rho (ρ)-dependent vs. ρ-independent
How does transcription
terminate?
Intrinsic
terminators
A Rho-dependent
terminator has a
sequence rich in C
and poor in G
preceding the
actual site(s) of
termination.
• ρ-Dependent Termination
• A hairpin forms
• if protein called ρis present, polymerase
detaches
• Dissociation is somehow coupled to ATP
hydrolysis
Rho factor pursues
RNA polymerase
along the RNA and
can cause termination
when it catches the
enzyme pausing at a
rho-dependent
terminator.
Transcription in Eukaryotes
• RNA polymerase I transcribes rRNA
• RNA polymerase II transcribes mRNA
• RNA polymerase III transcribes tRNA and
other small RNAs.
Animal RNA Polymerases
• Animal DNA-dependent RNA Polymerases
• Class
α-amantin sensitivity
Major Products
• I
Insensitive
rRNA
• II
Low Conc. (1-10 nM)
hnRNA
• III
High conc.
tRNA, 5S RNA
and small RNAs
• All have in common 2 large subunits and a number of smaller
subunits, as well as being zinc metalloenzymes.
What is the promoter difference between
prokaryote and eukaryote genes?
Enhancer
– Enhancers Work Upstream, Downstream
or in the Middle of a Gene
– They also work forwards or backwards
– Possible ways of working
Different transcription factors
Order of binding (differing concentrations)
Affinity of transcription factors
Elements combination in
type II Promoters
TATA box is a septamer (TATAAAA ) at 25 and is involved in positioning the
enzyme for correct initiation.
Initiator has a general form Py2ANT/APy2
and has the simplest possible form
recognizable by RNA polymerase II.
GC box is at -90 contains the sequence
GGGCGG and is recognized by the factor
SP1.
CAAT box (CCAATCT) is at –75 and is
recognized by a large group of
transcription factors and plays a strong
role in determining the efficiency of the
promoter.
Enhancer; Dehancer; Silencer;
Upstream Activating Sequences (UAS)
How to determine
promoter elements
• Change promoter sequence
• Promoter elements are defined by
mutations and footprinting
Have you ever wondered how your
genome works? Well, thanks to scientists
like Roger Kornberg, awarded the 2006
Nobel prize for Chemistry, who has
painstakingly studied the micromechanics
of transcription, we’re getting a much
clearer picture of what happens inside the
nucleus, and how the billions of metres of
DNA in your body are converted into RNA
by an enzyme called RNA polymerase II.
- from the announcement of 2006 Nobel prize for chemistry
More than 10 Subunits to form RNA
polymerase II
How does RNA polymerase II
initiate transcription?
• TBP: TATA-binding protein
• TAFs: TBP-associated factors
• TFIID protects a region extending farther
upstream
• TFIIA activates TBP by relieving the
TFIID
repression that is caused by the TAFs
• TFIIB binds adjacent to TBP and TATA
box
• TFIIF consists of two subunits. The
larger subunit has an ATP-dependent DNA
helicase activity and the small one contacts
the core polymerase.
• TFIIE and TFIIH are required for
promoter clearance to allow RNA
polymerase to commence movement away
from the promoter.
• TFIIH has several activities,
including an ATPase, a helicase,
and a kinase activity that can
phosphorylate the CTD tail of
RNA polymerase II; it is also
involved in repair of damage to
DNA.
Phosphorylation of the CTD
by the kinase activity of TFIIH
may be needed to release
RNA polymerase to start
transcription.
Most of the TFII factors are released before RNA
polymerase II leaves the promoter.
TBP is a universal factor
DNA-binding and activating functions in a transcription
factor may comprise independent domains of the protein.
What are Transcription units for RNA polymerase I ?
How many promoters in type III gene ?
• Two types promoters
upstream promoter and internal promoter
• upstream promoter: U6 snRNA
• Internal promoter: 5S RNA and tRNA
How does initiate in type III gene with polymerase III ?
5S RNA
tRNA
Termination of Eukaryotic
transcription
• Type II genes: Transcription stops after
AATAAA-Polyadenylation signal.
• Type I genes:3-4 consecutive Ts
• Type III genes: Stop after synthesis of
serial Us.