Saturday, 22 December 2012

RNAP Structure and function

X-ray crystal structure of RNAP from Archaea by Hirata
A simplified version of ekaryotic RANPII. Consists of an RNAPII-like enzyme and 2 TFs, TBP and eukaryotic TFIIB orthologue TFB.

RNAP common core consists of 5 subunits which are conserved from bacteria to humans. Bacterial RNAP is simplest. It consists of β'βαIαIIω subunits In eukaryotes RNAPII has additional polypeptides to form a 12 subunits complex.

Hirata crystallised RNAP form S. solfataricus.  RNAP resembles a crab claw with a portruding stalk formed by E'/F subcomplex. Relative positioning of RNAP core and stalk are highly conserved. It is proposed that function of the stalk  in RNAPII is to modulate clamp conformation. Without subcomplex, RNAPII clamp is in open conformation. In presence of subcomplex, clamp is closed.  In archaeal RNAP it is proposed that subcomplex facilitate transcription bubble formation of RNAP-promoter DNA complex under certain conditions.

a, Crystal structure (3.4 Å resolution) of the S. solfataricus RNAP. Each subunit is denoted by a unique colour (see surface representations inFig. 2 for colour-code and subunit–subunit interaction). The disordered clamp head domain is indicated as a dotted line. b, Crystal structure (1.76 Å resolution) of the D/L subcomplex (red, D subunit; yellow, L subunit). Domain organization is shown. c, Close-up view of the 4Fe–4S cluster-binding domain (residues 167–222 of the D subunit). Electron density calculated using the Fe-anomalous signal is shown in black mesh (sigma cutoff = 5).
Conserved structures around active centre and DNA-binding channel.
Surface representations of multi-subunit cellular RNAP structures from Bacteria (left, T. aquaticus core enzyme30), Archaea (centre, S. solfataricus) and Eukarya (right, Saccharomyces cerevisiae RNAPII13). Each subunit is denoted by a unique colour and labelled. Orthologous subunits are depicted by the same colour

Crystal structure of Thermus Aquaticus core RNAP at 3.3 angstrom resolution by Zhang
X-ray crystal structure shows a crab-claw molecule with a 27 angstrom wide internal channel. Mg2+ is on back wall of channel. It is chelated by a conserved motif.

β and β' make extensive critical interactions with each other. A major interface between them is at base of channel where active centre Mg2+ is chelated.

Substrate and inhibitor binding.
RNAP has binding sites for 2 NTP substrates: the i site, which will become 5' end of RNA transcript and i+1 site (elongation site) which will exteng the i site nucleotide in 3' direction where phosphodiester bond forms.

Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 angstrom resolution by Gnatt
Clamp closure
Clamp swings over cleft during transcription complex formation. This traps template and transcript. Clamp rotates by 30°. Most of clamp moves as a rigid body. But 5 switch regions undergo conformational changes and folding transitions.

Switches 1, 2, 4, and 5 form base of clamp. In free Pol II, 1 and 2 are poorly ordered and 3 is disordered. All 3 switches become well ordered in transcribing complex.  Conformational changes are accompanied by changes in salt linkage networks to bridge helix.

Downstream DNA mobility
Downstream DNA contacts Rpb5 jaw at a loop containing Pro118 and passes between Rpb2 lobe and Rpb1 clamp head.

Transcription bubble.
Downstream edge of bubble lies between poorly ordered downstream dsDNA and first ordered nucleotide of template strand at position +4, 3 nt before beginning of RNA-DNA hybrid.  Template strand follows a path along bottom of clamp and over bridge helix. Bridge helix residues Ala832, and Thr 831 position coding nt through vdW interactions.

Rbp fork loop 2 may maintain downstream edge of bubble. Rbp2 fork loop1 may help maintain bubble further upstream.
Figure 4
Maintenance of the transcription bubble. (A) Schematic representation of nucleic acids in the transcribing complex. Solid ribbons represent nucleic acid backbones from the crystal structure. Dashed lines indicate possible paths of nucleic acids not present in the structure. (B) Protein elements proposed to be involved in maintaining the transcription bubble. Protein elements from Rpb1 and Rpb2 are shown in silver and gold, respectively.

DNA-RNA hybrid
Base in template strand position +1 forms 1st of 9 bp of DNA-RNA hybrid between bridge helix and Rpb2 wall.  Electron density for hybrid is strongest in downstream region around active centre. This indicates high degree of order, important for high fidelity of transription. Further upstream density for DNA template strand reminas strong but RNA strand is weaker. This shows a loss in number of RNA-protein cntacts. Template DNA strand is bound by protein over entire length. RNA contacts are only downstrea, 5 upstream ribonts are hel through bp with template DNA.

Rpb1 and RPb2 make contacts to downstream and upstream parts of hybrid. Caused by ordering of Rpb switches 1, 2 and 3 on nucleic acid binding.

RNA synthesis
Active site metal ion location is suitable to bind phosphate between nt at 3'end of RNA and adj nt, +1 and -1 respectively.

Ribose sugar and DNA-RNA hybrid helix may cause specificity for ribo- rather than deoxyribonucleotides. 2'-OH of a ribont in substrate binding site may H bond ribose sugar and thus discriminate it from dNTP. Nucleic acid binding site is complementary to hybrid heli conformation and not DNA double helix conformation.

Positions -1 and -5 in transcribing complex can recognise RNA. by H bonding and discrimination. This contributes to specificity of RNA synthesis through proofreading. Presence of dNTP or incorrect base will destabilise.   Previously correctly synthesised RNA will be in hybrid region and RNA with misincorporated nt extruded at 3' end in a back-tracked complex. Extruded RNA is removed by cleavage at active site by TFIIS.

vdW contacts to nt base at end of hybrid region at +1 are by The831 and Ala 832 from Tpb1 bridge helix.

Protein-RNA contacts are important. NTPs are held in positions +1 and -1 for synthesis of first phosphodiester bond. After translocation to positions -1 and -2 dinucleotide product must be held by protein-RNA contacts. RNA is exposed at -4 and beyond.

RNA exit
Abortive cycling yields 2 or 3 residue transcripts up to 10. When transcript reaches 10, newly synthesised RNA must separate from DNA-RNA hybrid and enter an exit channel on surface. It is protected from nuclease attack. 3 loops extending from clamp. rudder, lip and zipper may play roles in hybrid dissocation, RNA exit and maintenance of upstream end of bubble.

Continuation of RNA path leads beneath lid. RNA extends to exit groove 1. Lid may maintain separation or RNA and template DNA. Zipper may have similar role.

Structural basis of transcription: RNA polymerases II at 2.8 angstrom resolution by Cramer
Surface charge of Pol II is almost entirely negative except for a uniformly positively charged lining of cleft, active centre, wall and saddle between clamp and wall. Positive charge of cleft may localise DNA. Positive charge on saddle may serve as exit path for RNA.
Figure 6
Surface charge distribution and factor binding sites. The surface of Pol II is colored according to the electrostatic surface potential (84), with negative, neutral, and positive charges shown in red, white, and blue, respectively. The active site is marked by a pink sphere. The asterisk indicates the location of the conserved start of a fragment ofE. coli RNA polymerase subunit β′ that has been cross-linked to an extruded RNA 3′ end
In single subunit structures Mg2+ ion. metal A coordinates 3'-OH group at growing end of RNA and α-phosphate of substrate NTP. Metal B coordinates all 3 phosphate groups of triphosphate. Both metals stabilise transition state during phosphodiester bond formation.

Diffusion of nucleoside triphosphates and role of entry site to RNAPII active centre
NTPs diffuse to active centre of RNAPII through a funnel-shaped opening that narrows to a negatively charged pore. Backtracked transcript is sequestered. Transcript is protected from nucleases. It is accessible only to TFIIS or GreA/B, which bind funnel surface and have a long slender protrusion that can reach up through pore to active centre. NTPs are delivered from entry site (E site) to addition site (A site) at 3' end of transcript  All NTPs bind E site. But only an NTP that can bp with DNA template can bind A site.

Antibacterial peptide microcin J25 inhibits transcripition by binding and obstructing RNA polymeras secondary channel by Mukhopadhyay
MccJ25 binds within RNAP secondary channel. Binding of MccJ25 within channel obstructs RNA seconary channel.  It inhibits transcription by interfering with NTP uptake. IT acts like a cork in a bottle.

It has also been shown that transcript- cleavage factors GreA, GreB and TFIIS must enter secondary channel to access active centre.  Binding of MccJ25 may block function of transcript cleavage factors, as binding competition occurs between Cy3-MccJ25 and GreB.

Intial transcription by RNA polymerase proceeds through a DNA-scrunchung mechanism by Kapanidis
Mechanism by which RNAP active centre translocates relative to DNA in initial transcription is contrersial. 3 models have been proposed.

First model is transient excursion. 2nd model is inchworming. 3rd model is scrunching. Experiments show that scrunching model occurs.
Fig. 2.
Initial transcription does not involve transient excursions. (A) Experiment documenting movement of the RNAP leading edge relative to downstream DNA [tetramethylrhodamine as donor at σ70 residue 366 (located in σR2, the σ70 domain responsible for recognition of the promoter –10 element); Cy5 as acceptor at DNA position +20]. (Top left) Structural model of RPo (28) showing positions of donor (green circle) and acceptor (red square). RNAP core is in gray; σ70 is in yellow; the DNA template and nontemplate strands are in red and pink, respectively. (Top right) E* histograms for RPo and RPitc, ≤7. The vertical line and vertical dashed line mark mean E* values for RPo and RPitc,≤7, respectively. (Bottom) Predictions of the three models. (B) Experiment documenting absence of movement of the RNAP trailing edge relative to downstream DNA [tetramethylrhodamine as donor at σ70residue 569 (located in σR4, the σ70 domain responsible for recognition of the promoter –35 element); Cy5 as acceptor at DNA position –39]. Subpanels as in (A).

Scrunching invokes a flexible element in DNA. In each cycle of abortive initiation RNAP pulls downstream DNA into itself, pulling  1bp per phosphodiester bond formed. It accommodates accumulated DNA as single-stranded bulges within unwound region. On release of abortive RNA, RNAP extrudes accumulated DNA, which regenerates initial state.
Fig. 4.
Initial transcription involves scrunching. (A) Experiment documenting contraction of DNA between positions –15 and +15 [Cy3B as donor at DNA position –15; Alexa647 as acceptor at DNA position +15]. Subpanels as in Fig. 2A. [The two donor-acceptor species in the E* histograms comprise free DNA (lower-E* species) and RPo or RPitc,≤7 (higher-E* species; higher FRET attributable to RNAP-induced DNA bending)]. Free DNA is present in all experiments, arising from dissociation of nonspecific complexes after heparin challenge during preparation of RPo, but is detected only in this experiment, because DNA contains both donor and acceptor only in this experiment. (B) Summary of results. Structural model of RPo (28) showing all donor-acceptor distances monitored in this work (Figs. 2 to 4A and figs. S2 to S8). Distances that remain unchanged on transition from RPo to RPitc,≤7 are indicated with thin blue lines. Distances that decrease on transition from RPo to RPitc,≤7 are indicated with thick blue lines. The red and pink arrows show the proposed positions at which scrunched templatestrand DNA and scrunched nontemplate-strand DNA, respectively, emerge from RNAP (i.e., near template-strand positions –9 to –10 and near nontemplate-strand positions –5 to –6).
FRET was monitored between a fluorescent donor and acceptor incorporated at specific sites in RNAP and DNA.  Results show that RNAP leading edge translocated relative to downstream DNA has scrunching or contraction of DNA segment between -10/-35 spacer DNA and downstream DNA.

Scrunching occurs only within DNA segment containing positions -15 to +15. Insufficient space in RNAP active centre cleft to accommodate scrunched DNA. Scrunched DNA must emerge from RNAP into bulk solvent immediately upstream of RNAP active centre cleft.

Initial transcription involves scrunching. Processive transcription elongation involved simple elongation, not scrunching.

It is suggested that a stressed intermediate exists in initial transcription. It has accumulated DNA-scrunching stress. They suggest that accumulated DNA-scrunching stress in intermediate provides driving force for promoter escape and productive initiation.

Stress is resolved either by
1. releasing RNA product, retaining interactions with promoter DNA, retaining interactions with initiation factors , retaining  an unchanged position of RNAP trailing edge, extruding scrunched DNA and reforming RP0 (abortive initiation)
2. retaining RNA product, breaking interactions with promoter DNA, breaking interactions with initiation factors, translocating RNAP trailing edge and forming RDe (promoter escape and productive initiation)

Structural basis of transcription: separation of RNA from DNA by RNAPII by Westover
Paths of RNA and DNA diverged beginning at position -8 . RNA-DNA hybrid was 8 bp long.

3 protein loops are lid, rudder and fork loop 1. They are important in RNA-DNA strand separation.

Lid is a wedge to drive RNA and DNA apart and interacts with residues -8,-9 and -10 of RNA. It forms a barrier to maintain separation and guide RNA to exit path.

Rudder is not directly involved in strand separation. It interacts with DNA at positions -9 and -10, preventing reassociation with RNA.

Fork loop 1 projects from Rpb2 and interacts with RNA at positions -5, -6 and -7 in hybrid region. It may delmit region of RNA-DNA strand separation, preventing unwinding of hybgrid past position -8. It extends region of protein contact with RNA-DNA hybrid from first 3 residues to entire hybrid.
Fig. 2.
Separation of RNA transcript from DNA template: the loop/strand network. (A) Portion of Fig. 1A, from residues –2 to –10, viewed from the front of the transcribing complex (rotated 90° around the RNA-DNA hybrid helix axis in Fig. 1A). Unpaired bases are colored orange (–8), purple (–9), and gray (–10). (B) Close-up of residues –7 to –10 of the model in (A). Average distances (in angstrom) between groups ordinarily involved in hydrogen bonding between complementary bases are shown. (C) Electron density for protein loops involved in strand separation. Backbone models of fork loop 1 (orange), rudder (green), and lid (purple) are fitted to electron density as in Fig. 1A. RNA and DNA models are from Fig. 2A. (D) Some residues of protein loops (carbon atoms, yellow; nitrogen atoms, blue) interacting with RNA and DNA. Fork loop 1 (Rpb2) residues Lys471 and Arg476, rudder (Rpb1) residues Ser318 and Arg320, and lid (Rpb1) residue Phe252 are shown.

3 protein loops interact also with one another. Lid interacts with protein elements to form an arch over the saddle. RNA exits active centre region through exit pore beneath arch. DNA exits above arch, preventing reassociation.
Fig. 3.
Exit path of RNA from the transcribing complex. (A) The transcribing complex with Pol II in surface representation, viewed down the axis of the RNA-DNA hybrid [from above the hybrid helix in Fig. 1; this is the “top” view shown in figure 1 of (10)]. Fork loop 1 is in orange, rudder is in green, lid is in magenta, and the RNA backbone is in yellow. Locations of the “clamp” and “wall,” important landmarks in the Pol II structure, are indicated by dashed lines. (B) The transcribing-complex structure sectioned and viewed as indicated by the dashed line and arrow in (A) to reveal the “arch” of protein density above the “saddle” and the “exit pore” through which RNA (yellow backbone) passes, following separation from the template DNA. (C) Two possible RNA exit paths from Pol II. The viewis the same as in (A), colored according to the electrostatic surface potential (negative in red, neutral in white, and positive in blue), with the Pol II wall, rudder, and lid removed to better reveal the sugar-phosphate backbone of the RNA spiraling upward from the active site. The two paths along which the RNA may be extended are the positively charged grooves, indicated by dashed yellow lines, labeled 1 and 2.
CTD domain (Wikipedia)

C-terminal domain (CTD)

RNA Pol II in action, showing the CTD extension to the C-terminal of POLR2A.
The carboxy-terminal domain (CTD) of RNA polymerase II is that portion of the polymerase that is involved in the initiation of DNA transcription, the capping of the RNA transcript, and attachment to the spliceosome for RNA splicing.[12] The CTD typically consists of up to 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser.[13] The carboxy-terminal repeat domain (CTD) is essential for life. Cells containing only RNAPII with none or only up to one-third of its repeats are inviable.[14]
The CTD is an extension appended to the C terminus of RPB1, the largest subunit of RNA polymerase II. It serves as a flexible bindingscaffold for numerous nuclear factors, determined by the phosphorylation patterns on the CTD repeats. Each repeat contains an evolutionary conserved and repeated heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which is subjected to reversible phosphorylations during each transcription cycle.[15] This domain is inherently unstructured yet evolutionarily conserved, and in eukaryotes it comprises from 25 to 52 tandem copies of the consensus repeat heptad.[14] As the CTD is frequently not required for general transcription factor(GTF)-mediated initiation and RNA synthesis, it does not form a part of the catalytic essence of RNAPII, but performs other functions.[15]

[edit]CTD phosphorylation

RNAPII can exist in two forms: RNAPII0, with a highly phosphorylated CTD, and RNAPIIA, with a nonphosphorylated CTD.[15]Phosphorylation occurs principally on Ser2 and Ser5 of the repeats, although these positions are not equivalent. The phosphorylation state changes as RNAPII progresses through the transcription cycle: The initiating RNAPII is form IIA, and the elongating enzyme is form II0. While RNAPII0 does consist of RNAPs with hyperphosphorylated CTDs, the pattern of phosphorylation on individual CTDs can vary due to differential phosphorylation of Ser2 versus Ser5 residues and/or to differential phosphorylation of repeats along the length of the CTD.[15] The PCTD (phosphoCTD of an RNAPII0) physically links pre-mRNA processing to transcription by tethering processing factors to elongating RNAPII, e.g., 5′-end capping, 3′-end cleavage, andpolyadenylation.[15]
Ser5 phosphorylation (Ser5PO4) near the 5′ ends of genes depends principally on the kinase activity of TFIIH (Kin28 in yeastCDK7 in metazoans).[15] The transcription factor TFIIH is a kinase and will hyperphosphorylate the CTD of RNAP, and in doing so, causes the RNAP complex to move away from the initiation site. Subsequent to the action of TFIIH kinase, Ser2 residues are phosphorylated by CTDK-I in yeast (CDK9 kinase in metazoans). Ctk1 (CDK9) acts in complement to phosphorylation of serine 5 and is, thus, seen in middle to late elongation.
CDK8 and cyclin C (CCNC) are components of the RNA polymerase II holoenzyme that phosphorylate the carboxyl-terminal domain (CTD). CDK8 regulates transcription by targeting theCDK7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH), thereby providing a link between the mediator and the basal transcription machinery.[16]
The gene CTDP1 encodes a phosphatase that interacts with the carboxy-terminus of transcription initiation factor TFIIF, a transcription factor that regulates elongation as well as initiation by RNA polymerase II.[17]
Also involved in the phosphorylation and regulation of the RPB1 CTD is cyclin T1 (CCNT1).[18] Cyclin T1 tightly associates and forms a complex with CDK9 kinase, both of which are involved in thephosphorylation and regulation.
ATP + [DNA-directed RNA polymerase II] <=> ADP + [DNA-directed RNA polymerase II] phosphate : catalyzed by CDK9 EC
TFIIF and FCP1 cooperate for RNAPII recycling. FCP1, the CTD phosphatase, interacts with RNA polymerase II. Transcription is regulated by the state of phosphorylation of a heptapeptide repeat.[19] The nonphosphorylated form, RNAPIIA, is recruited to the initiation complex, whereas the elongating polymerase is found with RNAPII0. RNAPII cycles during transcription. CTD phosphatase activity is regulated by two GTFs (TFIIF and TFIIB). The large subunit of TFIIF (RAP74) stimulates the CTD phosphatase activity, whereas TFIIB inhibits TFIIF-mediated stimulation. Dephosphorylation of the CTD alters the migration of the largest subunit of RNAPII (RPB1).

[edit]5' Capping

The carboxy-terminal domain is also the binding site of the cap-synthesizing and cap-binding complex. In eukaryotes, after transcription of the 5' end of an RNA transcript, the cap-synthesizing complex on the CTD will remove the gamma-phosphate from the 5'-phosphate and attach a GMP, forming a 5',5'-triphosphate linkage. The synthesizing complex falls off and the cap then binds to the cap-binding complex (CBC), which is bound to the CTD.
The 5'cap of eukaryotic RNA transcripts is important for binding of the mRNA transcript to the ribosome during translation, to the CTD of RNAP, and prevents RNA degradation.

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