Mechanisms of transcription By ZhaoYi 生物学基地班

 After having discussed the maintenance of genome, we now turn to the question of how that genetic material is expressed.

 Transcription is somewhat similar to DNA replication. Both involve enzymes that synthesize a new strand of nucleic acid complementary to a DNA template strand.

The differences are: 1>.RNA polymerase it does not need a primer; rather, it can initiate transcription de novo.

RNA polymerase

 DNA-dependent RNA polymerases are responsible for building RNA transcripts (mRNA, tRNA, rRNA) complementary to template strands of double stranded DNA, and regulation of their activity is often the final step in cellular pathways that control the expression of genes.

RNA Polymerase Structure  The massive holoenzyme contains 6 subunits: the θ subunit, β' subunit, β subunit, ω subunit, and two α dimer subunits.  however, the θ subunit is not a member of the core enzyme.

The holoenzyme

The core enzyme    ’’ 

Overall, the shape of RNA polymerase resembles a crab claw. The active site is found at the base of the pincers within a region called the “active center cleft”.

Active center cleft

2>.The RNA product does not remain base- paired to the template DNA strand----rather, the enzyme displaces the growing chain only a few nucleotides behind where each ribonucleotide is added.

3>.Transcription, though very accurate, is less accurate than replication of DNA. This difference reflects the lack of extensive proofreading mechanisms for transcripts from a single gene in a short time.

A series of steps for transcription  Initiation  Elongation  Termination

Initiation A promoter if the sequence that initially binds the RNA polymerase. As the replication, the DNA around the point where transcription will start unwinds, and the base pairs are disrupted.

Transcription always occurs in a 5’ to 3’ direction. Only one DNA strand acts as the template on which RNA is built. The choice of promoter determines which stretch is transcribed at which regulation is imposed.

Binding (closed complex) Promoter “ melting ” (open complex) Initial transcription

Elongation Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shift into the elongation phase. This transition requires further comformational Changes in polymerase that lead it to grip the Template more firmly.

Termination  RNA polymerase stop and release the product  In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.

Three defined step of initiation  Closed complex  Open complex  Stable ternary complex

Closed complex  The initial polymerase binding to the promoter  DNA remains double stranded  The enzyme is bound to one face of the helix

Open complex  the DNA strand separate over a distance of ~14 bp (-11 to +3 ) around the start site (+1 site)  Replication bubble forms

Stable ternary complex  The enzyme escapes from the promoter  The transition to the elongation phase  Stable ternary complex =DNA +RNA + enzyme

Bacterial promoter  σ 70 is the factor of E.coli RNA polymerase that has two conserved sequences, which are called consensus sequence. They are called -35 and -10 region.  Very few pomoters have this exact sequence, but most differ form it only by a few nucleotides.

Another class of s70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

The σ 70 factor comprises four regions called s region 1 to s region 4.  Region 4 recognizes -35 element  Region 2 recognizes -10 element  Region 3 recognizes the extended –10 element

One helix inserts into the DNA major groove interacting with the bases at the –35 region. The other helix lies across the top of the groove, contacting the DNA backbone

Interaction with –10 is more elaborate ( 精 细 ) and less understood  The -10 region is within DNA melting region  The  helix recognizing –10 can interacts with bases on the non-template strand to stabilize the melted DNA

UP-element is recognized by a carboxyl terminal domain of the  -subunit (  CTD), but not by  factor

Transition to the open complex  Structure changes open the DNA double helix to reveal the template and nontemplate strands. This melting occurs between positions -11 and +3, in relation to the transcription start site

 For s70 –containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement)

The striking structural change in the polymerase  1. the  and ’ pincers down tightly on the downstream DNA  2. A major shift occurs in the N-terminal region of  (region 1.1) shifts. In the closed complex,  region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft

Transcription needs: The initiating NTP (usually an A) is placed in the active site The initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme

The elongating polymerase is a processive machine that synthesizes and proofreads RNA

Synthesizing by RNA polymerase  DNA enters the polymerase between the pincers  Strand separation in the catalytic cleft  NTP addition  RNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time)  DNA strand annealing in behind

Proofreading by RNA polymerase  Pyrohosphorolytic （焦磷酸键解） editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi.  Hydrolytic （水解） editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimulation factor

Transcription is terminated by signals within the RNA sequence Terminator are the sequences that trigger the elongation polymerase to dissociate from the DNA There are two type of terminator: Rho-independent and Rho-dependent

Rho-independent terminator  contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs

Weakest base pairing: A:U make the dissociation easier

Rho -dependent terminators  Have less well-characterized RNA elements, and requires Rho protein for termination  Rho is a ring-shaped single-stranded RNA binding protein, like SSB  Rho binding can wrest the RNA from the polymerase-template complex using the energy from ATP hydrolysis  Rho binds to rut (  utilization) RNA sites  Rho does not bind the translating RNA

Transcription in eukaryotes  Eukaryotes have different RNA polymerases while bacteria have only one.  Several initiation factors ate required for efficient and promoter-specific initiation. These are called general transcription factors (GTFs)

RNA polymerase II core promoters are made up of combinations of 4 different sequence elements Eukaryotic core promoter (~40 nt): the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro

 TFIIB recognition element (BRE)  The TATA element/box  Initiator (Inr)  The downstream promoter element (DPE)

RNA polymerase form a pre- initiation complex with general transcription factor at the promoter

1. TBP in TFIID binds to the TATA box 2. TFIIA and TFIIB are recruited with TFIIB binding to the BRE 3. RNA Pol II-TFIIF complex is then recruited 4. TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex 5. Promoter melting using energy from ATP hydrolysis by TFIIH ) 6. Promoter escapes after the phosphorylation of the CTD tail

TBP binds to and distorts DNA using a sheet inserted into the minor groove A:T base pairs (TATA box) are favored because they are more readily distorted to allow initial opening of the minor groove

The other GTFs also have specific roles in initiation  ~ 10 TAFs: (1) two of them bind DNA elements at the promoter (Inr and DPE); (2) several are histone-like TAFs and might bind to DNA similar to that histone does; (3) one regulates the binding of TBP to DNA

TFIIB: (1) a single polypeptide chain, (2) asymmetric binding to TBP and the promoter DNA (BRE), (3)bridging TBP and the polymerase, (4) the N-terminal inserting in the RNA exit channel resembles the s3.2.

 TFIIF: (1) a two subunit factor, (2) binding of Pol II- TFIIF stabilizes the DNA-TBP-TFIIB complex, which is required for the followed factor binding  TFIIE: recruits and regulates TFIIH  TFIIH: (1) controls the ATP-dependent transition of the pre-initiation complex to the open complex, (2) contains 9 subunits and is the largest GTF; two functions as ATPase and one is protein kinase. (3) important for promoter melting and escape. (4) ATPase functions in nucleotide mismatch repair, called transcription-coupled repair.

in vivo, transcription initiation requires additional proteins including the mediator complex

Mediator  includes more than 20 subunits  Organized in modules

A new set of factors stimulate pol elongation and proofreading Transition from the initiation to elongation involves the Pol II enzyme shedding most of its initiation factors (GTF and mediators) and recruiting other factors

 (1) Elongation factors: factors that stimulate elongation, such as TFIIS and hSPT5.  (2) RNA processing (RNA 加工 ) factors  Recruited to the C-terminal tail of the CTD of RNAP II to phosphorylate the tail for elongation stimulation, proofreading, and RNA processing like splicing and polyadenylation.

elongation factors  P-TEFb: phosphorylates CTD Activates hSPT5 Activates TAT-SF1  TFIIS: Stimulates the overall rate of elongation by resolving the polymerase pausing Proofreading

Elongation polymerase is associated with a new set of protein factors RNA processing: Capping of the 5’ end of the RNA Splicing of the introns (most complicated) Poly adenylation of the 3’ end

Function of poly(A) tail  Increased mRNA stability  Increased translational efficiency  Splicing of last intron

Function of 5´cap  Protection from degradation  Increased translational efficiency  Transport to cytoplasm  Splicing of first intron

Other processes  Splicing: joining the protein coding sequences  3’ end polyadenylation

 RNA Pol I & III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP

That’s all, thank you!