What is transcription?How transcription works? Stages Machinery Molecular mechanism How transcription is regulated? Regulators Mechanisms Examples of transcriptional regulation Phage strategy RNA silencing

Molecular Biology (3/30~4/25, 2007)

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:

shentl@ntu.edu.tw

Ch. 9

Ch. 10,11

Ch. 12, 11

Prokaryotic Cell Eukaryotic Cell

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 vs. Eukaryotes

Transcription in prokaryotes

The basis of life

What is transcription?

☆ Gene Expression: Transcription

☆ Gene functions (majority) are expressed as the proteins they encode: Translation

Central Dogma of Biology: DNA → RNA → protein

Transcription = DNA → RNA

Translation = RNA → protein

RNA is structurally similar to DNA But……..

Gene Transcription: DNA → RNA

genetic information flows from DNA to RNA

Four stages of transcription:

1. Promoter recognition and initial melting (binary complex formation)

2. Initiation (ternary complex formation)3. Elongation4. Termination

RNA is identical in sequence with one strand of the DNA (but T→U), called coding strand.

by RNA polymerase

Transcription Unit

May include more than one gene

5’

,

3’

no number 0 mRNA

A transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene (particularly in prokaryotes).

Transcription unit

RNA polymerase

binding release

A relative location on a linear sequence

5’ 3’

(Primary transcript)

How transcription works?

Basic principles of transcription

Template recognition: polymerase and duplex

DNA

Initiation: polymerase* and promoters

Elongation: RNA polymerase

Termination: terminator

abortiveinitiation

Initiation

• Binding of an RNA polymerase to the dsDNA

• (Slide) to find the promoter

• Unwind the DNA helix

• Synthesis of the RNA strand at the start site (initiation site), this position called position +1

Transcription Bubble

To fulfill the principle process of transcription, that is

complementary base pairing, a transient bubble has 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.

Unwinding and Rewind DNANTPs polymerized to a RNA chainMoving in the DNA

Transcription Bubble

RNA-DNA hybrid length

~ 8 to 9 bases, it is short and transient

Ternary Complex:

Polymerase-DNA-RNA

Function of RNA Polymerase

About 25-base RNA molecule associated withthe ternary complex at any moment.

Progression of transcription bubble is

association with RNA polymerase

movement on DNA

DNA unwind aheadDNA rewind behind

5’ 3’

RNA

RNA extension

Elongation

1. Sliding:2. inchworm

Movement models

Direction 5’ to 3’

SubstratesATP, UTP, GTP, CTP

Phosphateα,β,γ

Nucleotide

Ribose5C -- 1,2,3,4, 5

RNA polymerization

Reaction in Transcription (RNA polymerization)

~40 nt/sec

Protein translationN → C termini ~15 aa/sec

DNA replication

5’ → 3’ ~800 bp/sec

α

β

γ

β

γ

α

3

23

NTP

NTP

NTP

NTP

5

4 1

5

23

5

4

5

3

Stages of transcription

: closed complex

: open complex

Promoter

Terminator

Abortive initiation: to ensure the initiation in a right way.(before the 10th base is added on nascent RNA chain within the bubble)

(5’)

(3’)

Extending RNA chain is accomplished with RNA poly (bubble)moves along DNA.

The bases after 9th enable added on the growing RNA chain.

move

Promoter clearsBubble moves on

Recognize termination signalRelease RNA chain (by disrupt RNA:DNA hybrid)

Dissociation of RNA pol

1. Sliding: common2. inchworm

Movement models

Binary

Ternary

Machinery in transcription

Prokaryotes have a single RNA polymerase enzyme--synthesizes mRNAs, rRNAs, and tRNAs

RNA Pol I rRNA

RNA Pol II mRNA

RNA Pol III tRNA, 5S rRNA

Transcription in Prokaryotes

Eukaryotes have three RNA polymerase Enzymes:

RNA polymerase

Transcribe over > 1000 transcription units. The complexity is modified byinteracting with diverse regulatory factors.

E. coli RNA polymerase

Both initiation & elongation

Initiation only

36.5 KD

36.5 KD

151 KD

155 KD

11 KD

70 KD

465kD

Core enzyme + sigma factor = holoenzymeRNA polymerase binds to the promoter

Structure and functions of E. coli RNA Polymerase

 2 subunits Enzyme assembly, Promoter recognition, factor binding

subunit Catalytic Center

' subunit Catalytic Center Template-binding

subunit Promoter specificity

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: α subunit

• Two identical subunits in the core enzyme • Encoded by the rpoA gene • Required for core protein assembly• May play a role in promoter recognition and reg

ulatory factors interaction• ADP-ribosylation on an arginine upon T4 infecti

on

1. Encoded by rpoB gene.

2. The catalytic center of the RNA polymerase• Rifampicin (used for anti-tuberculosis): bind to the β

subunit (12A away from active site), and inhibit transcription initiation. Blocking the path for extending RNA chain beyond 2-3 nts. Mutation in rpoB gene can result in rifampicin resistance.

• Streptolydigins ： resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.

3. subunit may contain two domains responsible for transcription initiation and elongation

E. coli polymerase: subunit

1. Encoded by the rpoC gene .

2. Binds two Zn 2+ /Mg 2+ ions and may participate in the catalytic function of the polymerase

• Heparin:binds to the ’ subunit and inhibits transcription in vitro due to it competes with DNA for binding to the polymerase.

3. ’ subunit may be responsible for binding to the template DNA .

E. coli polymerase: ’ subunit

1. Many prokaryotes contain multiple factors to recognize different promoters. The most common factor in E. coli is 70. (differential specificity)

2. Binding of the factor converts the core RNA pol into the holoenzyme.3. factor is critical in promoter recognition, by decreasing the affinity of

the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter

4. factor is released from the RNA pol after initiation (RNA chain is 8-9 nt)

5. Less amount of factor is required in cells than that of the other subunits of the RNA pol.

E. coli polymerase: factor

    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.

(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 on promoter recognition

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

reversible

Faster

Wide range

Fastest

Slow

Tight binding

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

recycled

Immediately after initiation

Evidence:1/3 of sigma factors are not associated with core enzyme while elongation

Molecular structure of RNA polymerasesin functioning

Architecture of RNA polymerases (prokaryotes)

T7 RNA polymerase Bacterial RNA polymerase (465kD)(<100 kD)

2α+β+β’+(σ)

~200 nts/sec

Further crystal structure will provide more direct and detailed view in a molecular level.

Specificity recognition between enzyme and DNA bases (upstream of startpoint +1)

Multiple subunits:

A channel/groove on the surface ~25A wide forms a path for DNA.

Path holds for 16 bp in prokaryotes 25 bp in eukaryotesMore DNA bp can reside on the enzyme

25A wide

~40nts/sec

Enzyme movement

Architecture of RNA polymerases (eukaryotes)

A channel/groove on the surfaceforms a path for DNA.Cleft between two

large subunits formsas an active center

Yeast RNA polymerase contains 12 subunits (10 are shown here)Nevertheless, it shares similar organization as bacterial one.

25 bp DNA can be held in the path.

Ternary Complex

Active center+

Enzyme movement

Channel within RNA polymerase

DNA in and out

DNA in

DNA out

DNA turns

RNA dissociated

How many bp(s) in the bubble?

rudder

RNA flipped out

Rigid straight duplex DNA

Flexible ss D

NA

(control by bridge protein)

entry

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

Change in conformation of “bridge” protein is closely related to translocation of the enzyme along the nucleic acid.

straight

bent

nt enters, adds, and interacts withthe bridge protein

nt still interacts with the bridge protein, which leads the protein to bending due to Pol moves one bp forward.

Meanwhile, bridge blocksfree nt enters.

Finally, bridge releases Its interaction with newlyadded nt on RNA chain.

straight

How does RNA polymerase find promoter sequences?

Random walk Directed walk vs.(Direct displacement)

Random diffusion

No DNA protein is known to work in this way RNA polymerase found promoters is very faster. Diffusion in the whole genome cannot support this fast.

Enzyme moves preferentially from a weak site to a strong site

Transitions in shape and size of RNA polymerase during transcription

Covered DNA length

75-80 bp(-55 to +20)

60 bp(-35 to +20s)

30-40 bp(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

Accessory factors are needed such as:GreA and GreB for E. coli RNA polymeraseTFIIS for eukaryotic RNA polymerase II

To correct mispositioned template during stall

* cleavage activity is from RNA polymerase itself.

(Create a 3’-OH for further polymerization)

One more function of RNA polymerase:

unwindRewindDNA/RNA bindingpolymerize RNA

A constant distance between active site and frond end

Sequence elements in Transcription

PromoterCoding sequenceTerminator

What is a promoter?

•The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction.

•Its structure (not transcribed) is the signal (others are needed to be converted into RNAs or proteins).

It is a cis-acting site.

•Different from sequences whose role is to be transcribed or translated.

What signal (structure) of a promoter provides?

(i.e. the distance of separation between -10 and -35; intermediate sequence is irrelevant)

(Closed binary complex formation)

(Open binary complex formation)

(recognition domain

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).

AT has only 2 H-bonds, which is easier to be broken

The sequence comparison of five E. coli promoters

Consensus TTGACA TATAAT

the most common base sequence to appear at such points on the DNA helix; there may be variations in various organisms

Consensus:

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

-10 unwinding domain: due to A-T pairs need lower energy to disrupt (melt)

Sequence around the startpoint (+1 to +30): influences the initiation event.

Open binary complex formation

Closed binary complex formation

Rate of promoter clearance

Other ancillary proteins may help RNA polymerase to recognize deficient promoters.

A-T rich sequence

Other structures may exist in a promoter

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

100-fold variationin vitro

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.

RNA polymerase-promoter interactions

J Bacteriol, June 1998, p. 3019-3025, Vol. 180, No. 12

-10 sequence (Pribnow box)• 6 bp sequence which is centered at around the –10

position (Pribnow, 1975).• A consensus sequence of TATAAT• The first two bases(TA) and the final T are most highly

conserved among other E. coli promoters• This hexamer is separated by 5 to 9 bp from position

+1, and the distance is critical• DNA unwinding is initiated at promoter by the

polymerase

-35 sequence: enhances recognition and interaction with the

polymerase factor • A conserved hexamer sequence around position

–35• A consensus sequence of TTGACA• The first three positions (TTG) are the most

conserved among E. coli promoters.• Separated by 16-18 bp from the –10 box in 90% of

all promoters

Transcription start site

• A purine (A or G) in 90% of all genes

• Often, there are C and T bases on either side of the start site nucleotide (i.e. CGT or CAT)

The sequence around the start site influences initiation

Promoter efficiency (1)

• There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold .

• The –35 sequence, -10 sequence, and sequence around the start sites all influence initiation efficiency.

Promoter efficiency (2)• The sequence of the first 30 bases to be

transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength .

• Strand separation in the initiation reaction

• Some promoter sequence are not strong enough to initiate transcription under normal condition, activating factor is required for initiation. For example, Lac promoter Plac requires cAMP receptor protein (CRP )

DNA unwinding • Necessary to unwind the DNA so that the

antisense strand to become accessible for base pairing, carried out by the polymerase.

• Negative supercoiling enhances the transcription of many genes but not all (e.g. gyrase) by facilitating unwinding .

• The initial unwinding of the DNA results in formation of an open complex with the polymerase and this process is referred to as tight binding

Supercoiling during transcription

∵ Supercoiled structure requires less free energy for the initial melting of DNA ∴ it enhances the efficiency of transcription in vitro

At initiation

After initiation 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)

recognition

contact

initiation

Touch down

extension

Sigma factor controls promoter recognitionDifferent sigma is used for distinct responses

The specificity is determined by recognizing different consensus sequences in promoters

"housekeeping"

starvation/stationary

flagellar sigma factor

nitrogen-limitation

extracytoplasmic stress

Bacillus subtilis sigma factors

factor gene function

43 rpoD, sigA housekeeping

28 sigD flagella/chemotaxis

29 spoIIGB, sigE sporulation

30 spo0H, sigH sporulation

32 sigC ?

37 sigB ?

spoIIAC spoIIAC sporulation

? spoIIIC sporulation

gp28 SPO1 28 phage middle

gp33/34 SPO1 33,34 phage late

Sigma factors may be organized into cascades

A new sigma factor displaces the previous sigma factor

Sigma factors directly contact DNA

conformation change

Release N-terminal autoinhibition due conformation changevia interaction with RNA polymerase

which contributes the binding specificities of sigma factors

Coding stand

melting

2.3

15A

20A

Free Holo: inside the active siteComplex: displace from active site

(most conserved)

2.4 4.2

domain1

Transcription initiation

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.

rate-limiting for the synthesis of productive RNAs

abortive RNA synthesis occurs

What is the role of sigma factor in abortive initiation/promoter clearance (escape)?

Elongation

Elongation

• Add ribonucleotides to the 3’-end (OH group)

• The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’ （ E. coli: 40 nt/sec)

• The enzyme itself moves in 3’ to 5’ along the antisense DNA strand.

RNA chain elongation• σFactor is released to form a ternary

complex of the pol-DNA-RNA (newly synthesized), causing the polymerase to progress along the DNA (promoter clearance)

• Transcription bubble (unwound DNA region, ~ 17 bp) moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear

• 3’ part of RNA forms hybrid helix (ca. 12bp) with antisense DNA strand.

• The E. coli polymerase moves at an average rate of ~ 40 nt per sec, depending on the local DNA sequence.

Termination

• The dissociation of the transcription complex from the template strand and separation of RNA strand from DNA

• Occurring at the terminator (often stem-loop or hairpin structure), some need rho protein as accessory factor.

it is a regulatory event

Hence, it is possible to readthrough the terminator (anti-termination) in a signal-dependent manner.

RNA chain termination• Termination: dissociation of RNA > re-annealing of DNA > release of

RNA pol

• Terminator sequence (stop signal): • RNA hairpin very common• Accessory rho protein

The DNA sequences required for termination arelocated 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

Rho-dependant termination

• Some genes contain terminator sequences requiring an accessory factor,the rho protein (ρ) to mediated transcription termination

• Rho binds to specific sites in the single-stranded RNA

• Rho hydrolyses ATP and moves along the nascent RNA towards the transcription complex then enables the polymerase to terminate transcription

rich

poor

A bias sequence preceding actual terminator site (RNA) is important for termination efficiency(rho dependent terminator).

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@ others

~275kD