Molecular Biology (3/30~4/25, 2007). What is transcription ? How transcription works ? Stages Machinery Molecular mechanism How transcription is regulated ? Regulators Mechanisms Examples of transcriptional regulation Phage strategy RNA silencing. Ch. 9. Ch. 10,11.
Molecular Biology (3/30~4/25, 2007)
What is transcription?
How transcription works?
How transcription is regulated?
Examples of transcriptional regulation
Ch. 12, 11
沈湯龍 (Tang-Long Shen) 助理教授細胞生物學一號館315室Tel: 3366-4998; E-mail: [email protected]
Transcription in Prokaryotes vs. Eukaryotes
Because there is no nucleus to separate the processes of transcription and translation, when bacterial genes are transcribed, their transcripts can immediately be translated.
Transcription and translation are spatially and temporally separated in eukaryotic cells; that is, transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.
Transcription in prokaryotes
What is transcription?
Central Dogma of Biology:
DNA → RNA → protein
☆ Gene Expression: Transcription
Transcription = DNA → RNA
☆Gene functions (majority) are expressed as the proteins they encode: Translation
Translation = RNA → protein
RNA is structurally similar to DNA
Gene Transcription: DNA → RNA
genetic information flows from DNA to RNA
by RNA polymerase
RNA is identical in sequence with one strand of the DNA (but T→U), called coding strand.
May include more than one gene
A transcription unitis the distance between sites of initiation and termination by RNA polymerase; may include more than one gene (particularly in prokaryotes).
no number 0
A relative location
on a linear sequence
How transcription works?
Template recognition: polymerase and duplex
Initiation: polymerase* and promoters
Elongation: RNA polymerase
To fulfill the principle process of transcription, that is
complementary base pairing, a transient bubblehas to be created.
Two strands of DNA are separated
(about 12~14 bp in length).
Template strand is used to synthesize
a complementary sequence of RNA.
The length of RNA-DNA hybrid
within the bubble is about 8~9 bp.
As RNA polymerase moves along the DNA, the transient bubble moves along with and the RNA chain grows continuously.
RNA-DNA hybrid length
~ 8 to 9 bases, it is short and transient
Function of RNA Polymerase
Unwinding and Rewind DNA
NTPs polymerized to a RNA chain
Moving in the DNA
About 25-base RNA molecule associated with
the ternary complex at any moment.
Progression of transcription bubble is association with
RNA polymerase movement on DNA
DNA rewind behind
DNA unwind ahead
Reaction in Transcription (RNA polymerization)
5’ → 3’ ~800 bp/sec
Direction 5’ to 3’
ATP, UTP, GTP, CTP
5C -- 1,2,3,4, 5
N → C termini ~15 aa/sec
Stages of transcription
: closed complex
: open complex
Bubble moves on
Abortive initiation: to ensure the initiation in a right way.
(before the 10th base is added on nascent RNA chain within the bubble)
Extending RNA chain is accomplished with RNA poly (bubble)
moves along DNA.
The bases after 9th enable added on the growing RNA chain.
Recognize termination signal
Release RNA chain (by disrupt RNA:DNA hybrid)
Dissociation of RNA pol
Machinery in transcription
RNA Pol I
RNA Pol II
RNA Pol III
tRNA, 5S rRNA
Transcription in Prokaryotes
Prokaryotes have a single RNA polymerase
enzyme--synthesizes mRNAs, rRNAs, and tRNAs
Transcribe over > 1000 transcription units. The complexity is modified by
interacting with diverse regulatory factors.
Eukaryotes have three RNA polymerase Enzymes:
RNA polymerase binds to the promoter
Core enzyme + sigma factor = holoenzyme
Both initiation & elongation
2 a subunits
Enzyme assembly, Promoter recognition, factor binding
Structure and functions of E. coli RNA Polymerase
Eubacteria RNA polymerase (Pol)
About 7000 RNA polymerase molecules are
present in an E. coli cell.
Most of them are engaged in transcription.
In a short period of time, 2000-5000 Pol molecules
can be synthesized.
E. coli polymerase: b subunit
3.b subunit may contain two domains responsible for transcription initiation and elongation
E. coli polymerase: b’ subunit
3.b’ subunit may be responsible for binding to the template DNA .
E. coli polymerase: s factor
Holoenzyme on promoter recognition
(Core enzyme + sigma factor = holoenzyme)
Core enzyme has the ability to synthesize RNA on a DNA template,
but cannot initiate transcription at the proper sites.
Holoenzyme has ~104-fold lower affinity for loose binding
complexes than core. About 60 min half-life reduce to <1 sec.
Holoenzyme has ~103-fold higher affinity for specific binding
to promoters than core with a half life of several hours.
Totally, sigma factor can result in
107 increase in DNA binding specificity.
Core enzyme does not distinguish between
promoters and other sequences of DNA.
Sigma factor is required only for initiation
Less than 10 bases
Beyond 10 bases leads to elongation
Recycle of sigma factor for the utilization of core enzyme
Sigma factor is much less in number than core enzyme
1/3 of sigma factors are not associated with
core enzyme while elongation
Immediately after initiation
Molecular structure of RNA polymerases
Architecture of RNA polymerases (prokaryotes)
Bacterial RNA polymerase (465kD)
T7 RNA polymerase
Specificity recognition between enzyme and
DNA bases (upstream of startpoint +1)
A channel/groove on the surface ~25A wide
forms a path for DNA.
Path holds for 16 bp in prokaryotes
25 bp in eukaryotes
More DNA bp can reside on the enzyme
Further crystal structure will provide more direct and detailed view in a molecular level.
Architecture of RNA polymerases (eukaryotes)
Yeast RNA polymerase contains 12 subunits (10 are shown here)
Nevertheless, it shares similar organization as bacterial one.
A channel/groove on the surface
forms a path for DNA.
Cleft between two
large subunits forms
as an active center
25 bp DNA can be held in the path.
Channel within RNA polymerase
DNA in and out
RNA flipped out
Flexible ss DNA
Rigid straight duplex DNA
(control by bridge protein)
How many bp(s) in the bubble?
Contact among the ternary structure in the active site
These contacts can stabilize the single strand nucleic acid chains.
Cycle of making and breaking bonds between enzyme and nucleic acids
nt enters, adds,
and interacts with
the bridge protein
nt still interacts with the
bridge protein, which
leads the protein to
bending due to Pol
moves one bp forward.
Meanwhile, bridge blocks
free nt enters.
Finally, bridge releases
Its interaction with newly
added nt on RNA chain.
Change in conformation of “bridge” protein is closely related
to translocation of the enzyme along the nucleic acid.
How does RNA polymerase find promoter sequences?
No DNA protein is known to work in this way
RNA polymerase found promoters is very faster.
Diffusion in the whole genome cannot support
Enzyme moves preferentially from a weak site to a strong site
Transitions in shape and size of RNA polymerase during transcription
Covered DNA length
(-55 to +20)
(-35 to +20s)
(interact w/ RNA pol)
How to resume the stalled/pausing RNA polymerase?
Cleavage 3’ end of RNA chain
Backtracks of RNA polymerase as a whole
(Create a 3’-OH for further polymerization)
A constant distance between active site and frond end
To correct mispositioned template during stall
Accessory factors are needed such as:
GreA and GreB for E. coli RNA polymerase
TFIIS for eukaryotic RNA polymerase II
One more function of RNA polymerase:
* cleavage activity is from RNA polymerase itself.
Sequence elements in Transcription
What is a promoter?
What signal (structure) of a promoter provides?
AT has only 2 H-bonds, which is easier to be broken
(Open binary complex formation)
(Closed binary complex formation)
(i.e. the distance of separation between -10 and -35;
intermediate sequence is irrelevant)
Pribnow, D.: Nucleotide Sequence of an RNA Polymerase Binding Site at an Early T7 Promoter. PNAS 72, 784 (1975).
Pribnow, D.: Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J. Mol. Biol. 99, 419 (1975).
Schaller, H. et al.: Nucleotide Sequence of an RNA Polymerase Binding Site from the DNA of Bacteriophage fd. PNAS 72, 737 (1975).
The sequence comparison of five E. coli promoters
the most common base sequence to appear at such points on the DNA helix;
there may be variationsin various organisms
Prokaryotic promoters display four conserved features:
1. Startpoint: >90% PURINE (A or G)
2. -10 consensus sequence (Pribnow box)--TAtAaT
T80 A95 t45 A60 a50 T96
3. -35 consensus sequence--TTGACa
T82 T84 G78 A65 C54 a45
4. Distance (spacing) between the -10 and -35 sequences
(The distance is critical in holding the two sites at the appropriate separation for
the geometry of RNA polymerase.)
5. UP element. TA rich sequence upstream of promoter.
Functions of promoter domains
-35 recognition domain
Closed binary complex formation
-10 unwinding domain: due to A-T pairs
need lower energy to disrupt (melt)
Open binary complex formation
Sequence around the startpoint (+1 to +30):
influences the initiation event.
Rate of promoter clearance
Other ancillary proteins may help RNA polymerase to recognize deficient promoters.
Other structures may exist in a promoter
A-T rich sequence
It interacts with the α subunit of the RNA polymerase,
which to ensure the higher gene expression.
Down mutation: mutations are tend to be concentrated in the most highly conserved positions.
Up mutation: less cases happen within promoters
RNA polymerase-promoter interactions
A promoter with consensus sequences for the -10 and -35 regions (boxed) is shown; the sequences of actual promoters deviate from those shown here.
The "jaws" of RNA polymerase are shown on the right of the molecule. This region of the RNA polymerase would grasp the DNA downstream of the catalytic site. Contacts between RNA polymerase and promoter DNA are shown by the solid lines. Not all contacts occur in every RNA polymerase-promoter interaction, but in all known cases (including promoters activated by regulator proteins), at a minimum, some contacts between and the 10 region appear to be required.
J Bacteriol, June 1998, p. 3019-3025, Vol. 180, No. 12
The sequence around the start site influences initiation
Supercoiling during transcription
∵ Supercoiled structure requires less free energy for the initial melting of DNA
∴ it enhances the efficiency of transcription in vitro
DNA is rotated during RNA pol movement; front is overwound and behind is released.
A twin domain on transcribing DNA formed
RNA polymerase binds to one face of DNA
(-9 to +3 for unwinding)
Sigma factor controls promoter recognition
Different sigma is used for distinct responses
flagellar sigma factor
The specificity is determined by recognizing different
consensus sequences in promoters
Sigma factors may be organized into cascades
A new sigma factor displaces the previous sigma factor
Sigma factors directly contact DNA
which contributes the binding specificities of sigma factors
via interaction with
Free Holo: inside the active site
Complex: displace from active site
DNA-dependent RNA polymerases are promoter binding,
DNA strand melting,
RNA chain initiation and
nascent RNA chain formation, and
finally escape from the promoter sequences.
abortive RNA synthesis occurs
rate-limiting for the synthesis of productive RNAs
What is the role of sigma factor in abortive initiation/promoter clearance (escape)?
it is a regulatory event
Hence, it is possible to readthrough the terminator (anti-termination)
in a signal-dependent manner.
RNA chain termination
The DNA sequences required for termination are
located prior to the terminator sequence.
Formation of a hairpin in the RNA may be necessary
RNA hairpin structure: an intrinsic terminator
near the base of the stem.
Hairpin leads RNA pol to slow/pause
The rU.dA RNA –DNA hybrid has an unusually weak base-paired structure;
it requires the least energy of any RNA-DNA hybrid to break the association
between the two strands.
A model for intrinsic termination
Termination efficiency determinants:
@ The Sequence of the hairpin
@ The length of the U-run
@ Sequences both upstream and downstream of the intrinsic terminator
@ Ancillary proteins
A bias sequence preceding actual terminator site (RNA) is important for termination efficiency
(rho dependent terminator).