test terms

what are recognize the sigma subunit of rna pol sigma 70

-35 box and -10 box are 2 core promoter elements that recognize the sigma subunit of rna pol sigma70

what are the 2 core promters

-35box and -10 box secequence TTGACA -10 BOX TATAAT

...ara operon

.2 Ara operon has 2 operator ARAO1 which contols the arac operon and ARa O2 control ara B ara A and araD it has CAP binding site for CAP-CAMP and negative regualtor regualtes through a represson loop..

2 models for PIC assembly test question

1.)sequential assembly 2.) pol II holenzyme both begin with TBP compoenent of TFIID binding to tata box in promoter sequential assembly a.)RNA polII TFIB + TBP RNA pol 2 TFIIF TFIIF TFIIE TFIIH b.) RNA pol II TFIIF FE TFIIB

What is the type of bond that forms between the 5' end of newly synthesized mRNA and the Cap? 4

5'>>>5' linkage or a 5' 5triphosphate bridge The starting point for capping with 7-methylguanylate is the unaltered 5′ end of an RNA molecule, which terminates at a triphosphate group. This features a final nucleotide followed by three phosphate groups attached to the 5′ carbon.[3] The capping process is initiated before the completion of transcription, as the nascent pre-mRNA is being synthesized

explain what are four types subunits alpha beta B'

a B B' and sigma gene rpoA gene product 2 alpha subnuits 40 KD each functions enzymes assembly promoter recognition binds some activators ropB B subnunit 155 KD function catalytic center ropc B' subunit 160 KD catalytic rpoD sigma subunit 32-90 kd - promoter specificity

Explain phosphrylation of the pol 2 CTD

CTD phsophorylation cycle TFIIH sunbunit kinase 28 phsophrylate serine 5 transcription elongation complex TEC is arrested at cheeckpoint for pre-mrna capping . P-TEFB ( postive transcription elongation factor ) PTEFB ( Burk 1/2 or Ctk1 in yeast) then phosphorylates seriine 2 which allows further elongation. specific phsophatase dephosphorylates the CTD at ser 5 overcomes promoter pausing

TEst Question Dna methylation in mammal consist of what

Critical for normal embroogensis /development gene regulation hypermethylation of CpG island promoter region is associated with gene silence chromatin compaction genome stability silencing endogenous viral sequence suppression of homologious recombination between repeats genome defense x chromosome inactivation females genomoic imprinting

Free sigma subunit does recognize single strand non-templante strand T or F

False free sigma subunit does not recognize single -strand non template REEMEMBER sigma specifically recognize -10 and -35 region and those with an UP stream promter element which is typically AT rich its the terminal domain of Aminio acid c terminal that regonize upstream regulator element sigma converts sequence specific binding to promoter recognize element -10 and -35

explain the CAP SITE along with the other sites of the lac operon

LAC I = upstream Cap binds just upstream of rna pol LAC I= stop laz downstream of dna Cap binding site which CAP/ CAMP to the laci Gene upstream of rna pol CAP site CRP- CAMP receptor protein promoter- protect by rna pol secequenc of lac operator overlapping with promoter lac z gene start

so what happens to lac operon in absence of lactose? understand the concept

Lac I consitituvely transcribe mrna for lac repressor which then translate to lac repressor proein which is a NEGATIVE REGULATOR which is sequence specific binding protein that specifically binds to operator dna sequences just dowinstream or overlapping the promoter sequence or lacoperon with binding or repressor protein summary no B-gal is syntheize since no lactose is available to be metabolize dna sequence of operator overlaps dna sequence of promoter binding of repressor to the operator physically interferes with binding of rna pol to the promoter

how can rna pol 1 and 2 and 3 can be distinguish explain

amanitit acitivy amancing 3 pol is resistant and 2 less resistant to 3

list the prokaryotic rna pol structure ? 5 points

dna double helix 3 end jaws in configuration ribonucleotide phosphate ribonuceloetide tunnel active site rna pol short region of dna /rna helix rna exi channel flaps in closed position dna rewinding lobster claw structure active site rna pol

explain the following defnitions promtores and promoter complex

promoter - dna sequence that binds rna pol to initiate transcription transcription initiation -synthesis of first phosphodiester bond in nascent RNA

6. List the Roles of the 5' CAP on mRNA.

protect rna from degradition the enhance transcription stability of mrna they enhance the transport of mrna from nucleus to cytoplasm they enhance effiencey of splicing

the basal promoter / PIC cycle

recruitment POL II IIF IIE IIB II D IIA med IIH med + promoter Dna>>>>> preinitation complex + ATP yeilds open comlex >>>> intitation NTP elongation RNA pol II IIB

List and explain 2 mechanisms of transcription termination in prokaryotes TEST QUESTIONs

rho independent and rho dependent termination rho independent also called intrinsic termination rho independent- intrinsic termination include pallindromic regions that form hairpins varying in length from 7 to 20bp the stem loop structue includes a G-C rich region and is followed by a run of U residences follwed by a run of U residues rho independent - IS DEPENDENT ON FORMATION OF STEM LOOP STRUCTURE IN THE RNA GC REGION IN THE STEM SINGLE STRAND U REGION - syntheize near the termination site base near the termination site structure interacts with rna pol cause it to pause during elongation pause couple with the weak bp of rU dA bases pairs at the termination site displaces of mrna chain signals rho dependent termination rho is protein cause depression in the ability of rna pol rho first binds to rna pol and waits for the syntehsis of the rho binding site to be formed . rho binds to ther rna at the roading loading site or rho utlization site rut and has ATPase activity that provides it energy move along rna chain 5 to 3 direction rna stalls in the termination region just after making the hairpin structure pulls up on the nascent rna strand snaps it off

what are the step taken for dna footprinting

1 purified rna pol 2 purified promoter sequenc 3. purified pol bind rna than on one end of 5 'end label radionucleotide so portion of dna is protected by rna pol which form a complex puried dna rna is tread with diexoidnuelodease 1 which make nicks use concentrated dnase 1 generated proximal 1 nick per dna molecule in solution dna is not protected with rna polymerase ever nucleotide cleavage of dnase however region bind rna pol will be protected from cleavage from dnase after dnase treatement purified dna denature strand run dna on high resolution dna sequence gel which generate ladder of dna sequence binds represenet dnase 1

what are stages of transcription initiation TEST question

1.) forming a closed promter complex 2. holenzyme can melt a short region of the dna at the promoter to open promoter complex which pol is bound tightly to the dna transcription initiate -dna unwinds 3 incorporating the first few nucleotide 4.) abortive initation transcripts not until it reach 10nt reaches promoter clearance genetic short aborptive transcript unstable until it reaches transcription intaition to promoter clearnece d.) promter clearance promoter proximal -near the promoter upstream -to left downstream to right transcription proceed from left to right as transcrtipition elongateds it forms a transcription buble

list the varies phases and step of rna pol that is bind to dna 10 points

1.) intation complex contasins sigma covers 75-80 bp promter dna that was determing by footprint 2.) intitatial elongation complex forms at 10bases loses sigma and loses contact in 35-55region after the intatial complex undergo s promoter clearance the intatil intation complex sigma loses its contex or protecton of dna posistion minus -35 and -55 General elongation comlex forms at 15-20 bases and covers 30-40bp 3. diagram as the elongation complex transition to a full elongation complex cover 30 to 40 bp of dna during process

TEST QUESTION consider e.coli cells having one of the follwoing mutations A mutant lac operator that cannot bind repressor A mutant repressor that cannot bind to lac operator a mutatant lac repressor that cannot bind to allolactose a mutant lac promoter region that cannot bind CAP plus cAMP

1.)Mutant operator Oc that makes it deffective in binding to represssor in conclusion a wild-type operon remain repressible but mutant operon is not it makes lac products even in the absence of lactose bc operon is connected to the mutant operators is affected this mutation is cis -dominaint 2. mutatnt repressor gene I- no lactose products in absences of lactose I+ wildtype repressor gene makes enough normal repressor to reprs both operons so mutation is recessive 3.) if repressor gene cannot bind to inducer-allolactose the mutant repressor therefore binds to irreversibly to both operators and renders both operons uninjucible this mutation is dominant 4.) cannot bind to operator having a wild type and mutant compose of mixture s heterotertramerss cannot bind to operator operon remains depress even in absence of o lactose bc the mutant protein posion the activity of the wild-type protein we call this mutation dominant negative

what is the lac operon and was is it function

3 genes that code forthe ability to carry out lactose fermentation Lacz beta galactosidase LacY: galactosidase permase LacA galactosidase transacetylase beta-galactisidase gene catalyse the conversion lactose into glucose + galactaose permase -facilitate lactose transport across the cell membrane transacetylase gene function is not clear may function cell detoxification continue on back of lac operon repressor in the following order i=regulator gene ( repressor) + promoter + operator+ z beta galactosidase + y(permese gene lactose transport accors the membreane + a ( transacyteylase) function i s trully unknown detoxification continue 3 regulator elements and genes LAC I + gene repressor LAC promoter= initaites transcription Lac operator- regulates dna transcription rna pol binds to promoter take rna pol holenzyme with sigma factor -10 to -35 reguglator sequence transcribes polycistronic mrna lac z lac y lac a POLYCSITRONIC MRNA = has 3 binding ribosmal site which ribosomal binds to beta galactosidase ribosomal binds to permease ribosomal binds transacetyalse which allow it to regulate 3 genes it a CATABOLIC operon its turn on in the presences of lactose and turn off in the absence of lactose operator - is a segement of dna which transcription factor binds to regulate gene expression by repression it operon -is a function unit of genmoic dna containig a cluster of genes under control of single promoter

explain the different types of chromatin remdeling complex

4 classes are distinugist by their ATPASE component SWI/SNF= switch sniff ISWI INO all four alter their structure of nucelosome core to make dna more accessible not only to transcription activators but also to nucelase and other protein swi/SNF complex in mammals have BRG1 and its atpase and 9-12 BRG1 associated factor SANT domain - another domain slide that appears in dna binding this help SWI/SNF bind to nucelosome members o ISWI calsses of remodeling comples have SANT domain Access to nucleosomal DNA is governed by two major classes of protein complexes: Covalent histone-modifying complexes. ATP-dependent chromatin remodeling complexes. Covalent histone-modifying complexes[edit] Specific protein complexes, known as histone-modifying complexes catalyze addition or removal of various chemical elements on histones. These enzymatic modifications include acetylation, methylation, phosphorylation, and ubiquitination and primarily occur at N-terminal histone tails. Such modifications affect the binding affinity between histones and DNA, and thus loosening or tightening the condensed DNA wrapped around histones, e.g., Methylation of specific lysine residues in H3 and H4 causes further condensation of DNA around histones, and thereby preventing binding of transcription factors to the DNA leading to gene repression. On contrary, histone acetylation relaxes chromatin condensation and exposes DNA for TF binding, leading to increase gene expression.[2] ATP-dependent chromatin remodeling[edit] Chromatin remodeling complexes in the dynamic regulation of transcription: In the presence of acetylated histones (HAT mediated) and absence of methylase (HMT) activity, chromatin is loosely packaged. Additional nucleosome repositioning by chromatin remodeler complex, SWI/SNF opens up DNA region where transcription machineray proteins, like RNA Pol II, transcription factors and co-activators bind to turn on gene transcription. In the absence of SWI/SNF, nucleosomes can not move farther and remain tightly aligned to one another. Additional methylation by HMT and deacetylation by HDAC proteins condenses DNA around histones and thus, make DNA unavailable for binding by RNA Pol II and other activators, leading to gene silencing. ATP-dependent chromatin-remodeling complexes regulate gene expression by either moving, ejecting or restructuring nucleosomes. These protein complexes have a common ATPase domain and energy from the hydrolysis of ATP allows these remodeling complexes to reposition (slide, twist or loop) nucleosomes along the DNA, expel histones away from DNA or facilitate exchange of histone variants, and thus creating nucleosome-free regions of DNA for gene activation.[12] Also, several remodelers have DNA-translocation activity to carry out specific remodeling tasks.[13] 1.) nucelosme slding 2.) remodeled nucelosome 3.) nucelosome displacement 4.) nucelosme replacement

You are studying a new species of frog that was discovered in the Amazon rainforest. How would test if the mRNA has a cap, describing the experiments and the results (draw)? 6

5 cap or cap first has roles cap is required for splicing CBP ( cap binding protein ) binds to 5' cap and facilitates transport of mrna out nuculeus into cytoplasm translation of mrna stimulates polyadenylation decapping involved in regualtion of mrna turnover cap might therefore exprect to protect the mrna from attack RNase that begin at the 5-endof their substrates and that cannot cleave triphosphates ANSWSER you would first test by using a label reovirus rna that is eiter cap with m7GPPG blocked with GpppG or uncapped then inject into frog eggs oocytes leave it in for about 8 hours then purfied them an anlyze them by glycerol gradient centrifugation reovirus must exist in 3 classes large medium and large which shown by sepeartion of centrifugation include with 5 end rna with the 5 end that sufferd degradiation clarify not cap structure

WHAT IS TRP ATTENUATOR MEDIATED REGUALTION EXPLAIN

ATTENUATION IMPOSES AN EXTRA LEVEL OF CONTROL ON AN OPERON OVER AND ABOVE THE REPRESSOR OPERATOR SYSTEM. IT OPERATES BY CAUSING PREMATURE TERMINIATION OF TRANSCRIPTION OF THE OPERON WHEN THE OPERONS PRODUCTS ARE ABUNDANT HIGH LEVELS TRP OPERON WANTS IT TO BE TURNED OFF BC SYNTHESIS EXCESS TRPY HIGH LEVEL DONT NEED ANYMORE RNA BEING SYNTHESIS THIS METHOD IS USED TRP OPERON REPRESSION IS WEAK AND TRP OPERON CAN STILL OCCUR IN THE PRESSECE OF REPRESSOR SO LOW TRPTOPHAN TRANSCRIPTION OF TRP STRUCURAL GENS RNA PL READS TRHOUGH THE ATTENUATOR SO THE STRUCTURAL GENES ARE TRANSCRIBED IN PRESENE OF HIGH TRYPTOPHAN THE ATTENUATOR CAUSE PREMATURE TERMINATION OF TRANSCRIPTION SO TRP GENES ARE NOT TRANSCRIP HIGH TRP LEVELS RNA TRANSCRPTS LEADER CODS FOR LEATER PEPTIDES PPPA UGG UGG= TRP UGG=TRP LEADER PEPETEDE LEADER PEPTIDE FROMS A FROMATION OF HAIRPIN LOOP AT POSTION CODON 3 AND 4 CODON 2 = RIBOSOME VERY SIMILAR TO RHO INDEPENDENT TERMINATION MRNA IS TRP EDCBA IS NOT SYNTHEIZE TRANSCRIPTION TERMINATIES AFTER SYNTHEISS OF LEADER RNA REASON BC THE INVERTED at REPAEAT FORMS HAIRPIN LOO[ lOW LEVELS IN SUMMARY RIBISOME TRANSLATING LEADER RNA INTO LEADER PEPTIDE MUST PAUSE BC TRP LEVELS ARE LOW PAUSING OF RIBOSOME OVER TRP CODIONS ALLOWS SEQUCE 2 AND 3 FORM HAIRPIN STRUCTURE NOW SEGMENT 3 CAN NOLONGER FORM A HAIRPIN STRUCTURE AND TRANCRIPTION TERMINATION IS NOT FOUND AND TRANSCRIIPTION POCEEDS

Describe the Torpedo Model for Transcription Termination of eurkaryotes

After the mRNA is completed and cleaved off at the poly-A signal sequence, the left-over (residual) RNA strand remains bound to the DNA template and the RNA polymerase II unit, continuing to be transcribed. After this cleavage, a so-called exonuclease binds to the residual RNA strand and removes the freshly transcribed nucleotides one at a time (also called 'degrading' the RNA), moving towards the bound RNA polymerase II. This exonuclease is XRN2 (5'-3' Exoribonuclease 2) in humans. This model proposes that XRN2 proceeds to degrade the uncapped residual RNA from 5' to 3' until it reaches the RNA pol II unit. This causes the exonuclease to 'push off' the RNA pol II unit as it moves past it, terminating the transcription while also cleaning up the residual RNA strand. Similar to Rho-dependent termination, XRN2 triggers the dissociation of RNA polymerase II by either pushing the polymerase off of the DNA template or pulling the template out of the RNA polymerase.[9] The mechanism by which this happens remains unclear, however, and has been challenged not to be the sole cause of the dissociation.[10] In order to protect the transcribed mRNA from degradation by the exonuclease, a 5' cap is added to the strand. This is a modified guanine added to the front of mRNA, which prevents the exonuclease from binding and degrading the RNA strand. A 3' poly(A) tail is added to the end of a mRNA strand for protection from

Enhancer you suspect that a sequence upstream of gene is acting as an enhancer design an experiment to show that the sequence acts an enhancer and not as a promoter

DNA element that binds one or more activator stimulates transcription of a gene or genes Enhancer are usually found upstream of the genes they influence , but they can also function if inverted or moved hundreds or even thousands of bp away non promoter elements that bind protein factos and stimulate transcription by defnition they act at distance aids in transcription of genes recognized by an auxillary factor silencer by contrast depress transcrption enhancer and silencers are position and orientation independent dna elementsthat stimulate or depress respectively the transcription of assoicated gene experiment would 5'flanking region of sv40 deletion mutations in this region depress transcription behavior suggested that 72bp constiuted another upstream promter element which it wasnt upsteram promoter element 72bp repeats still stimulate transcription even if it inverted or moved all the qay around to opposite side of circular sv40 genome over 2kb away promoter

explain the 2 mechanism by which dna methylation can silence genes

Deacytelation/ MEtylation MeCP2 binding protein binds to methylatined chromatin and recruits transcriptional repressor complex to deacylate and methylated TRanscription factor inhibiion dna is mehtylated such that methylation sensisitive transcritption factors can bind

what is the rate of transcription for euk and bacterial rna pol ?

EUk rna pol 2 15-30nt/sec bacterial pol 50-100 nt /sec

elongation , termination TEST QUESTION

Elongation 1.) rate of elongation with E.coli RNA pol= 40 nucleotides/sec ; T3 RNA pol= 200 nucleotides /sec 2.) E. coli RNA pol cover ( footprints) 28-35bp of during elongation 3.) Mechanism for overcoming stalled pol during elongation B.)Termination in prokaryotes and transcription is termination as elongation is completed specfic signals in dna trigger transcription terminator 1.) dependent on specific DNA sequences ( terminator) specific siganl in dna 2.) transcription complex dissociates and rna pol and nascent rna released 3.) Rho dependent termination a.) requires termination factor protein rho b.) mechanism not fully understood 4.) Rho independent termination- involves formation of tem loop secondary struncture encoded by dna template and formed by nascent RNA

what are CpG island how importan are they in gene relegation

GC rich regions of 500 bp often associated with promoters almost alway un methlated 70 percent of all CPGs are methylated in mammalian genomes 45ooo cpg island in the human genome dna methylation in mammals occur primarily within the seuence Cpg usually unmethylated CPg sequences

lIST AND EXPLAIN A PROKARYOTIC ENAHCER AND ENHANCER BINDING PROTEIN

GLN a- ECODES GLUTAMINE SYNTHESIES OF ENTERIC BACTERIA TRANSCRIBED BY SIGMAFACTOR RNA POL HOLENZYME INSTEAD OF OF MORE COMMON SIGMA 70 HOELNZYME ntrc - NITROGEN REGULATORY PROTEIN C OF ENTERIC BACTERA TRANSCRIPTIONAL ACTIVATOR OF GLnA gene Ntrc functions similar to eukaryotic enhancer binding proteins binds 2 specific dna sequence upstream 140 -180 of gln A promoteser still activates transcription when movied 1kb upstream or downstream of gln A promoter NTRC facilitareds conversion of glnA promter closed to open regulates glnA gene and other sigma 54 holoenzyme promoter by binding to distal dna sequence and inucing conformational change at GlA promoter closed >>>> open promoter complex

fill in the missiong blank writer reader eraser histone acteylase ? ? methyltrasferase ? ? ? ? ?

HAT HMT PRMTs= writers protein arginine methyly trasferase readers bromodomain chromodomain tudor domain ERaser HDACS KDMS= lysine demthylase Writers AC HAT ME HMTS P= kinase REaders AC bromo ME Chromomo , PHD tudor P 14-3-3 BRCT ERasers HDACS AC DMTS ME P phosphatase

TEST question 7 list explain promoters and promoter complexes give the definition of each promoter transcription initiation postion +1

Promoter- DNA sequence that binds rna pol to intiate transcription transcription initiation =-synthesis of first phosphdiester bond in nascent rna ( new for form dna) position + 1- position of nucleotide in dna template that encodes the first nucleotide of mrna

explain and list the mechanisms of bacterial transcription initiation test question 2 6 pts

RNA pol synthesize rna from one strand of a double strand dna template REMEMBER its may be true or false E.coli a single rna pol synthesize most if not all mra rRNA and TRNA 3.)RNA pol holenzyme composed 4 subunits a B B' and 0' core enzyme ( a B B') synthesize RNA randomly on ds DNA template sigma facter: confers promoter binding and specifcty 4.) sigma factor a.) most E.coli promters bound by sigma 70 -associated RNA pol b.) allows correct bindinb and transcription initiaition at specific promter c.) sigma is released upon transcription intiation ; core enzyme continues transcription elongation d.) ALternative sigma factors regonized different promters differen prokaryotes varies form sigma factor sigma factor has specificity for differ subset of prokaryotic promoter

TFII D major component and explain its function TEST Question

TBP -TATA binding protein TAFS - TBP associated factors also evidence for Cell type specific TBP TAF complexes witout TBP that can functionally replaces TFIID TFIID is the first protein to bind to DNA during formaion of the pre-initation transcription complex of RNA pol II. binding to TFIID to TATA box in the promoter region of gene initiates the recuritment of other factor required for RNA polII to begin transcription TFIID joins with TFIIA at the TATA box contains 14 subunits TBP associated factos TAFS pic assembly

4. Describe the process of 5' methyl capping

The 5' Cap and the Poly A Tail 5' End Capping Post-transcriptional processing of the 5' end of the RNA product of DNA transcription comes in the form of a process called the 5' cap. At the end of transcription, the 5' end of the RNA transcript contains a free triphosphate group since it was the first incorporated nucleotide in the chain. The capping process replaces the triphosphate group with another structure called the "cap". The cap is added by the enzyme guanyl transferase. This enzyme catalyzes the reaction between the 5' end of the RNA transcript and a guanine triphosphate (GTP) molecule. In the reaction, the beta phosphate of the RNA transcript displaces a pyrophosphate group at the 5' position of the GTP molecule. The cap is formed through a 5'-5' linkage between the two substrates such that the GTP molecule is oriented in the opposite direction as the other nucleotides in the RNA transcript chain. Once in place, the cap plays a role in the ribosomal recognition of messenger RNA during translation into a protein. Prokaryotes do not have a similar cap because they use other signals for recognition by the ribosome. The Poly A Tail Post-transcriptional RNA processing at the opposite end of the transcript comes in the form of a string of adenine bases attached to the end of the synthesized RNA chain. This string of adenine is called the "poly A tail". The addition of the adenines is catalyzed by the enzyme poly (A) polymerase, which recognizes the sequence AAUAAA as a signal for the addition. The reaction proceeds through mechanism similar to that used for the addition of nucleotides during transcription. The poly A tail is found on most, but not all, eukaryotic RNA transcripts. Its significance remains unknown.

True or False E.coli sigma factor recognize promters with different consensus sequences

True typical gene RPOD facto sigma 70 use general -35 sequences TTGACA separation 16-18bp -10 sequence TATAAT

what is the extended promter element

UP element from -60 to -40 and core promoter element -35 box to -10 box extended promter element= up element + core promter element core promoter elements -35 box -10 box unwound region transcription

what are UP promoter elements give a list of a gene that contains a UP element

UP element, a component of bacterial promoters located upstream of the −35 box hexamer, increases transcription by interacting with the RNA polymerase α-subunit. element found upstream of the core promoter in euka ribosomal RNA gene rrnB P1 promoter with UP promter element some prokaryotic have some prokaryotic promoters have additional sequences which are shown have ribosomal rna gene which have up element upstream in additional core promter -35 box and -10 box

typical prokaryotic promoter recognize by E.coli sigm 70 rna pol share important pol recognition sequence recognize all of the following except

a - 10 region ( pribnow box): TATAAT consensus sequence b.) -35 region :TTGACA consensus sequence hint remember consensus sequence - is the average of similar sequences if examine a number of sequence its likely to have a T as first c.) different promoter have similar but not identical -10 and -35 similar have a stronger promter d.) mutations with these regeions alter promoter strength and function e . distance between -10 and -35 region important variations go downstream ( distance between -10 and -35 important bc insertion or deletion of rna pol binding go down significantly strength of promoter mostly determined by the affinity of rna pol form promoter sequence g.) region unwound by pol appears to be between -9 and +3 includes right end of -10 seq and extending to just downstream of transcription of initation synthesis of RNA in 5 to 3 direction nucelotides added to 3 end from ribonucleotide precursor

TFIID

a class II general transcritpition factor that binds first to tata box containing promoter in vitro serves as a nucleation site around which the preiniation complex assembles. contains tata -box binding protein TBP and TBP-associated factors TAF1 + TAF2 + TAF9 + TAF6 TBP+ + TFIIB TFIIB - BRE TFIIB-BREu TBP-TATA TAF2-INR TA6-DCE

explain what is the ara operon and how does it works

ara operon is controlled by the Arac protein Ara C represses the operon by looping out the dna between the 2 sites araO2 and araI1 that are 210bp apart arabinose can depress the operon by causing ARAc to loosen the its attachment to araO2 bind araI2 instead this breaks loop and allows transcription of operon CAP and CAMP further stimulate transcription by binding to a site upstream of arai ARaC contriols its own syntheis by binding to arO1 and prevent leftward transcription of aRAc gene

WHAT ARE THE DEFINTIONS CATABOLITE REPRSESSION Camp CAP

catabolite repression of gene or operon by glucose is more ikely by a catabolite or breakdown producte of glucose CAP- a protein which together with cAMP a citeivates operons that are subject to catabolite repression basically lac operon in inactive state as long as glucose is present favor of glucose metabolism and against use of other energy sources contribute some break down of glucose metabolite known a catabolite repression CAP-CAMP- is postive control of lac operon so in summary CAP catabolite activtor protein postive control of lac operon and certain inducible operons code for sugar meatablize enzyme mediated by CAP CAP_AMP- stimulates transcription bc CAMP is depreessed by glucose this sugar prevents the stimulation of transcription therefore the lac operon is only stimulaated or activated when glucose concentraion is low need arise to metabolize an alternate enegy source CAP + CAMP allow formation of an open promoter complex when rna pol binds to dna containin a lac promoter randomly binds weakly to dna this binding is susceptible to inhibition when fifampicin is added along with nucleotides howeever when CAP and CAMP binds forms open promoter complex this is not suceptible to inhibition when rifampicin and nucleotides are added

what rna pol pausing / stalling during elongation

during process transcription and elongation they are certain sequences that cause pausing or staling of pol which has to be restarted some case stalling can cause cause by secondary structing rna being transcribe to form secondary structure cause stalling are pausing pol so they has to be mechanism MAjor steps for overcoming pausing stalling is first POLYMERSASE BAcK TRACk along dna pol goes back along dna goes back dna is the proces that moves backward the 3 end of nascent transcrpt take out of the active site is push outward pol as goes backward so the over hanging 3 end of NAcent transcript that occurs after back tracking is than leave of by specific nuclease that regenerate a 3 oh cleavage site right in active site give pol the opportuity stalling and grap the proces overcoming stallinga or pausing is involve in back tracking in pol cleaved of overhanging in 3 end of nascent rna and tepmp of elongation to process multple time if neccessar to overcome stalling or pausing of pol figure 9.7 a stalled rna pol can be released by cleaving the 3 end of transcrpt 1 usual postion of rna pol during elongation 2. rna pol stalled and back tracks 3.) region of rna is cleaved 4. now 3 end located on catalytic site 5. catalytic site resume elongation of rna

what heppens in the presence or absence of glucose with or w/out lacotse

e.coli prefers the use of glucose over other sugars for energy as a carbon source prensence of both glucose and lactose glucose is preferentially metabolize and proteins encoded by the lac operon are synthesize at very low levels therefore another regulator signal must be requireed to monitor glucose levels and activate the lac operon in addiction to inactivation of the lac repressor by allolactose CAMP= binds to regulator proteins called CAMP high glucose levels lower cAMP low glucose levels increase cAMP remember cAMP binds to CAP ( catabolite gene activator protein) and cAMP-CAP complex activates transcription of the lac operon binding to promoter region CAP alone w/out bound cAMP will not stimulate transciption CAP-cAMP bind to lac operon just upstream from RNA pol binding site in promoter CAP- cAMP binding to adjacent DNA sequence facilitates rna pol binding to promoter thereby stimulating rate of transcription initation

readers and erasers

eraser mechanism by which dna methylated erase or reverse very complicated proces riders -put methy groups on dna

TEST QUESTION explain the PIC assemble pathway order assembly

factors bind to a promoter in a step wise fashion 1 recruitment of TBP inititating evernt which TBP bindsto TFIIB>>>>>TFIIB reacts with TFIIF + CTD POLII TFIIF interacts with the RNA pol and is required for preventiong unspecific initiation at non -promoter sites as well as for effiecent transcription elongation TFIIE stimulates the helicase and ATP ase activities of TFIIH >>>>> transcription initation complex >>>>> NTP +ATP >>>>> ADP nascent RNA release of GTF except TBP and elognation pol II with phsophroylated CTD

trp operon is an inducible operon true or false

false its a negative control operon truly and repressible operon protein that inhibits gene transcription -In prokaryotes, this protein binds to the DNA in or near the promoter. when bound to operator, no production of tryptophan -too much tryptophan present, bounds to repressible operon, which then binds to operator

footprinting

finding a target dna sequence or bindig site of dna binding protein dnase footprinting is performed by binding the prtein to its end labled taget than attacking the dna-protein complex with DNASE when the resulting dna is electrophoresed the protein binding sit shows up as a gap or footprint in the pattern where the protein is protected the dna from degradation DMS follow similar principle

what are the 2 basal promter elements in higher euakrytoeis

focused transcription initation ( typically found in regualted promoters ) Dispersed transcription intiation commonly found in constiutive promters houskepping consitutively express genes are transcrbed from multiple start sites tissue specific and regulated genes are transcribed from a single start site transcription initation takes place at genes harboring specific basal promoter elements TATA INR BRE (TFIIB recongninton element) DPE downstream core promter elemeent

list and explain GTF

general transcription factors combine with rna pol to form preinitation complex that is competent to initiate trascription contains rna pol 2 and 6 generl transcription factors that bind to each other in specif order to ensure proper function TFIID joins with TFIIA at the tata box TFII B to the DA complex TFIIF help RNA pol II to -34 to 17 then TFIIE and TFII H to make the DABPOLFEH preiniation complex

so explain what happens in varies combination of lactose and glucose know its a conswquenc in lac operon regulation

glu glucose and lactose presenct cell prefer glucose dont use lactose the lac operon turn off -no cap binding bc CAMP levels or low and CAP wont bind a aresult you wont get a stable binding are very effiecnt binding of rna pol to lac operon and don regulate bc camp cannot bind bc high glucose and low CAMP levels what happens high glucose and low lactose BC HIGH GLUCOSE YOU ALSO DONT GE CAMP BINDING BC LOW CYCLIC AMP LEVELS AND CAP BY IT SELF DONT BINDING SO IT DONT FACILTIATE IN RNA POL LOW LACTOSE YOU ALSO GET REPRESSOR BIND BC CAP IS NOT BIND IN SUMMARY + GLUCOSE + LACTOSE OPERON IS OFF BC CAP NOT BOUND +GLUCOSE - LACTORSE OPERON IS OFF BOTH BCAUSE LAC REPRESSOR IS BOUND AND BC CAP NOT BOUND -GLUCOSE AND - LACTOSE OPERON OFF BC LAC REPRESSOR BOUND -GLUCOSE + LACTOSE OPERON - GLUCOSE AND + LACTOSE OPERON ON

what does the following denote H3k27me3 h3k3me3 h3kpac h3k27 H3K27me3 H3K9me3 H3K36me3 H3K9ac H3K27ac

histone modifications h3k27 repression h3kpme3 h3k27me3 =repression denotes a specific chemical modification of the DNA-packaging protein histone H3. This modification is commonly associated with repression of expression of nearby genes.[1] inactive genes H3K4me3 is enriched in transcriptionally active promoters.[12] H3K9me3 is found in constitutively repressed genes. H3K27me3 is found in facultatively repressed genes.[7] H3K36me3 is found in actively transcribed gene bodies. H3K9ac is found in actively transcribed promoters. H3K14ac is found in actively transcribed promoters. H3K27ac distinguishes active enhancers from poised enhancers. H3K122ac is enriched in poised promoters and also found in a different type of putative enhancer that lacks H3K27ac.

Describe the experiment by Hofer and Darnell and what the conclusion from that experiment was.

hnrna is a precursor of mrna they posses long chain of AMP residues known as poly A polyadenylation is not synomyous to transcrption termination they peform early work of polyA and polyadenylation they purfy hela cell poly from rest of mrna release it with 2 enzymes Rnase A which cuts pryimidine nucleotide C and U and RNASE T1 which cut G nucleotides basically they cut every RNA except A's after they electrophoresize to detremine size found coresponded size to be 100-200 found the average size to be 250 nt

all of the following are activated by NIFA protein except test question

homologue of NTRC catlyzes formation of open promoter complex bonds to specfic dna seqece upstream of nifh promter position 132 unlike ntrc and eukaryotic enhancer does not work downstream of promoter also works by forming loop between nifa binding site and polymerase promoter complex loop fromation assisted by IHF ( integration host factor ) proteins which nomds tp s[ecof dna sequenc between NIFA site and promoter and bends dna

e.coli cells is grown in absence of glucose which of follwing will be true

if lactose present lac mrna syntheisis will occur at high levels

so what happen to lac operon in presnence of lactose

lac I transcribe mrna >>>>>> which is a lac repressor >>>>>>> since lactose is presence lactose is an inducer known as allolactose which binds to repressor in a lock key formation >>>>> this result in interferces of the repressor protein binding to the operator downstream and result in transcription in lac z y a proteins in summary lac I reprssor stilll syntheiszie however the metabolite lactose called allolactose acts as in inducter binds to lac repressor alters conformational change so repressor an no longer bind to operator there alow RNA pol to bind to promoter and transcribe polycistronic mran that transcribe b-gal permease transacetylase protein so operator turn on metablize lactose by acting on b-gal

what are the prokaryotic transcriptional regulation

lac operon trp operon gln regulation by ntrc -enhance fuction IHF ingetration host factors

so what are some of strateges that one can use to identiy important regulator sequence for eukaryotest?

linker scanning analysis and promoter deletion analysis and enhancer analysis by transient expression assay linker scannin analysis of promoter of element do linker scanin mutation across a region of interest promoter deletion analysis test for the independedce location of promoter acitivity

what is intragenic and intergenic dna methylation

methylation of promoter regions with high cpG density is highyl correlated with gene silencing methylation in promoter regions of low cpg density not correlated with gene silencing intragenic non promoter dna methylation has been reported act as a postive regulator gene transcription regulate alternative promoter usage infulence nucelosome postion affet elongation regulate rna splicing intergenic dna methylation may regulate the function of long range regulatory region

mediator

multiprotein complex facilitate interactions between GTFs and actiator yeast coactivator that binds to an acitvator and helps it stimulate assembl of a preinitation complex general tanscroption facdor bc is part of most of not all preinitation complex

what are the similarties of trp operon and lac operon

negative control of trp operon is in sens the mirror image of negative control of lac operon lac operon resonds to inducer that causse the repressor to dissociate from the the operator depresses the operon trp operon resonds to repressor that includes corepresssor thyptophan which signals the cell that it has made enough of this amino acid corepressor binds to aporerpressor chaning its confromational so it can bind better to trp operator thereby repressing the operon

what is the nifH gene

nitrogen fixation gene in nitrogen fixing baceria also transcribe by sigma 54 holeenzyme function NIFA is not postion independ like NTRC and like eukaryotic enhancer activated by NIFA protein

give 5 examples of how the tryptophan operon is regulated

operon encodes 5 enzymes a-e therefore under condition fo high extracellular Trp operon turned off low extracellular trp operon turned on exact opposite to lac operon toe extracellular lactose lac operon encodes catabolic enzyme trp operon encodes syntehic enzyme trp operon has 2 simultaneous mode of transcription regulaion 1 repressor -mediated regulation and 2 attenuator mediated

desribe promter proximal pausing including all important proteins and phsophorylation events

pausing overcomse signalsllllllllllllllllllllllllllll tell genes to acitvate to reengage in full enlogation siganal receve another factor PTEFB has kinase phosphorylates serine 2 on CTD on rna pol2 as phosphorlateding NELF and DISF what that accomplosh dissociates NELF from enlongation complex DSIF to negative elgonation factor to postive elongation factor DSIF (DRB sensisty inducting factor) NELF negative elongation factor T-TEFb postive transcrition elongation factor b another leverl of gene expression overcoming promoter proximal complex RNA polymerase II (Pol II) temporarily stops transcription after synthesizing 30-50 bases, and resumes elongation only after stimulations by various signaling molecules and developmental cues. This phenomenon, called promoterproximal pausing, is observed in 10-50% of the entire genes from Drosophila embryos to human cells.

IPTG labeked synthetic inducer isopropylthiogalacside to reprssor O+ operator Z+ lac z lacY concenpt defintely test question in the table fill + b-galactasodase syntheisis and- for no beta galactosidase syntheisis glucose is absent in all cases genotype -IPTG +IPTG O+ Z+ Y+ Oc Z+Y+ O+Z-Y+ Oc Z+ Y-

phenotype all genes are wild type there fore wild-type function is present and b-gal is produce upon induction only phentotype minus inducer= IPTG = - IPTg phenotype plus inuducer= IPTG + IPTG Oc +Z Y+ Oc mutant allele is dominant over to the O+ allele the mutant operator is unuable to bind repressor protein and will therefore consititutively produce b-gal protein in its operon regardless of the presence f absence of inducer phenotype minu inducer= IPTG + phenotype plus inducer = IPTG + O+ Z- Y+ z mutant allele is recessive to the z+ allele the enzyme will produce by the wild type gene is sufficient to give wild type phenotyp if recessive z it will give I no phenotype for neither with or without inducer Oc Z+ Y- sinc oc mutant alles is dominant over to the o+ allel the mutant operator is unbable to bind to repressor protetin will consitutively produce b-gal protein in operon regardless of absece of inducer ITP phenotype minus indeucer - phenoty pluce inducer=+

phosphorylation is modification commonly found in which amino acids

phosphorylation found in serine and threonine acetyaltion lysine methylation lysine mon di tri mehtyaltion argione ubiquitaion lysine sumolyation lysine adp ribosylaion glutamate deimination argine > citrileine

when trp is presen in the environment of e.col the tryp binds to the repressor what are the rna pol for eukaryotics and what are the functions

pol 1 transcribe r RNA genes ( 28 s 18s 5.8s rna gnes) accounts 80-90 of total cellular rna large rna precursor pol transcribes protein encoding mrna and several non -protein encoding genes consist of consist 12 subunit that are conserve among divesre of eukaryotics mrna snrna mi rna pol 3 transcribe trna 5srna 7srna components of signal recognition particl genes and other small rna encoding genes include gens ofr rna splicing trna and 5s genes have internal promoter major differnce eukaryotes have 3 rna pol compare prokaryotes 1

TEST Question what is GTF and PIC

pre-inititation complex the combination of rna pol and general transcritiption factos assembled at a promoter its competent to initatie transcr general transcription factors eukaryotic protein that particpate alone with rna pol in froming pre-intation complex PIC assembles at he basal promoter region of agene tight binding involves formation of an open promoter complex in which the dna at the transcription start site has melt to allow the po II l to read it CLASS II factors pre-initation complex PIC -identified by invitro transcription assay on template of adenovius MLP major late promoter

what is repressor mediated regulation tryptophan

promoter operater attenuator attenuaton- a mechanism of transcription control that involves premature transcription termination attenuator= a region of dna upstream from one or more structural genes where preomature transcription termination ( attenuation ) can occur repressor -mediated regulation promoter=p operator=o attenuatedr lambda E=D=C=B=a IN HIGH TRP >>> MRNA MAKES TRP REPRESSOR PROTEIN ALONG WITH TRP CO REPRESSOR BINDS TOGETHER TO OPERON >>>> WHICH RESULT IN NO TRANSCRIPTION low TRP >>>> PRODUCE MRNA REPRESSOR CANNOT BIND TO OPERATOR IN ABSENCE OF TRP AND TRANCRIPTION IS INATITED

TEST question which model involves the formation of a stem loop secondary structure

rho independent termination-invovles formation of stem loop secondary structure encoded by dna template and fromed by nascent rna

what is the role of a promoter ?

rna pol sequence specific dna binding protein recognize specific sequence in dna associatated with promoters so rna can position down properl on bacterial promoter to initate transcription

how do bacterial rna polymerase interacte with promter during transcription initation ?

rna poly holenzyme recognize critical regonition sequence in the promoter region sigma specifically regonize -10 and -35 regulator regions than those for promoter region Transcription initiation : bacterial rna pol -promoter interaction C terminal domain alpha subunit regonize upstream regulator element =A+T rich up N terminal alpha connected to B and B' subuti and connected to alphac terminal REMEMBER sequence specific interaction with the promter via sigma subunit sigma in holenzyem regonize -10 element va base specific interation with the non termplate strand of promoter

test question explain the sigma cycle

rna polymerase binds to the promoter at left causing local melting of the dna . as the polymerase moves to the right elongating the rna the sigma factor dissocates and joins with a new pol to initate another rna chain

what is epiptope tagging how can it be uee to purify pol 2 from yeast

small foriegn eptipe attatch to a foreign yeast pol 2 subunit Rpb3 by engineering its gene after immunoprecipitation they separted the labeled poly peptides of precipated protein by sds-page and detected by radiograph add an ab directed against the epitpoe tag which immunoprecipated the whole rna pol separting from contaimined proteins this give very pur pol in just one step add strong detergent sds which separtes denature the subunit of the purfied pol electrophorese the denature subunit of pol

what is scrunching .what does the rna pol achieve by scrunching

so what happen to rna pol during early stages of abportive transcription 1 moverment of transcription excursion , inchworming and scrunching scrunching the rna pol dont mover really dna coils or compress inside promoter scrunching basically dna scrunchess in promoter no change shape or movement of pol actually move compasion or scrunching in rna pol scrunches is the model thats true FRET -inter molecular proximitify this address abortive intiation acceptor fluorphore donor flurophore to address aborptive ination link the acceptor fluorphore red to dan template and donor flourphore scrunching A hypotheis to explain abortive transcription that invokes an rna pol that squeezes more dna inoto itself without move relative to the dna SCRUNCHING is required for promoter clearence trailing edge upstream dna transicent excursion increase separtion inchwroming no chage scrunching no change leading edge promoter dna inchworming -increase separtion scrunching no change downstream and promoter dna = scrunching decrease separtation scrunching of model to fluorphores should come closer together you should se increase in effiecen ofFRET that what in fact no change of FRET for the others transicent excursion so they left scrunching most likely model when modeldirectly what they did scrunching is required for promoter clearance scrunching is like compressing spring

Explain the tanscription cycle

tata box -30 INR +1 and +30 DNA promoter tata xo TBP or TFIID +TFIIA >>>>>>>> TFIIB>>>>TFIIF RNA POL A>>>> TFIIE >>>TFIIH form PIC complex PIC closed >>>ATP dna melting >>>PIC open >>>> initiation CTD phosphorylation promoter clearnce elongation release of TFIIB AND E AND H

TBP-TATA

tbp is sddle shaped protein with two -fold symmetry recognize minor groove DNA is show bent through 80 at the TATA box

so How do alpha subunit potion of holeoenzyme recongnize the upstream promter element for those promoter that have regulator sequence

the a-subunit of rna pol purified alpha C terminal domain non template demonstrated by dna footprinting experiment purified a subunit template strand blank portion repression purified segmen f apha subunit DNASE 1 purified segement of alpha subunit rna pol bind to it DNA footprinting is is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells. The regulation of transcription has been studied extensively, and yet there is still much that is not known. detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. protected of dna in exact postion where its bound

Chip chromatin immunoprecipaiation assay what is the main purpose of a chip experiment and explain the basic steps

the main purpose of CHIP is characterize the binding patter of specific protein of entire genome A method of purifying chromatin cotaining protein of interest by immunoprecipating the chromatin with an ab directied against that protein of against an epitope tag attached to the proteins cHIP detects specific protein -DNA interactions in chromatin in vivo it uses an abs to preciptate particular protein in complex with DNA and PCR to determine whether the protiein binds near a particular gene 1ab binds to epitope attah to a protein of interest which in turn bound to a specfic site on ds dna 2.) identify the dna in a immunoprecipate 3.) primer specfic dna of interest used pcr to amply a portion of dna 4.) production of a dna fragement of correct predicited size indcates that the protein idid indeed bind to the dna of interest

How do cells make up for the fact that the triphosphate at the beginning of the mRNA makes it susceptible to degradation by RNases? 3

through 5' MRNA capping pppXpY >>>>> Rna triphosphatse>>>>> ppXPy >>>>> GPPP >> Guanlytransferase >>>>> m7Gppxpy>>>>>> methyltransferase >>>>m7GpppXmP7 One of the terminal phosphate groups is removed by RNA triphosphatase, leaving a bisphosphate group (i.e. 5'(ppN)[pN]n); GTP is added to the terminal bisphosphate by mRNA guanylyltransferase, losing a pyrophosphate from the GTP substrate in the process. This results in the 5′-5′ triphosphate linkage, producing 5'(Gp)(ppN)[pN]n; The 7-nitrogen of guanine is methylated by mRNA (guanine-N7-)-methyltransferase, with S-adenosyl-L-methionine being demethylated to produce S-adenosyl-L-homocysteine, resulting in 5'(m7Gp)(ppN)[pN]n (cap-0); Cap-adjacent modifications can occur, normally to the first and second nucleotides, producing up to 5'(m7Gp)(ppN*)(pN*)[pN]n (cap-1 and cap-2);[7][8] If the nearest cap-adjacent nucleotide is 2'-O-ribose methyl-adenosine (i.e. 5'(m7Gp)(ppAm)[pN]n), it can be further methylated at the N6 methyl position to form N6-methyladenosine, resulting in 5'(m7Gp)(ppm6Am)[pN]n.[3]

explain the transcription bubble

transcription takes place in a bubble which rna is syntheized by base pairing with ones strand in the transiently unwound region as the bubble progress the dna duplex reforms behind it displacing the rna in the form o a signel poly nucleotide

explain the process of Transcription TEST question1.)

transcription unit is a sequence of dna transcribed into a single rna starting at the promoter and ending at he terminator 1 pol recognize and bind to a specific sequence which is the promter promter ( starting point )<<< proximal >>>distial terminator upstream <<<<<<<< and downstream during transcription the bubble is maintained within bacterial rna pol which unwinds and rewinds dna , maintains the conditions of partern and template dna stand synthesis unwinding in frontal portion of dna in the bubble than you have a template strand synthezie rna form a dna/rna hybrid you have an uncoding strand of dna you have a rna binding site and you have your rewinding of dna

RNA pol that bind to dna can protect dna during varies phase during transcription and intaition

true

TRUE or FaLSE e.coli perfer to use glucose over other suguars including lactose for energy and carbons sourese

true

True or false strong evidence that ntrc functions by binding to enhancer and contacting rna pol via beinding and intervening dna once NTRC contact pol and induces open promter complex formation NTRC no longer required for remaining steps of transcription elongation termination not all prokaryotic enhancers function via loop formation late genes of bacteriaphage t4

true

similar strucute of rna in that has lobster claw shape similar in shape of pokaryotic true or false

true

what is epitope tagging

using genetic means to attach a small group of amino acids an epitope tag to a protein enable proteins to be purfied raidily by immunoprecipateion with ab that recognize epitpe tag

list and explain transcription terminator of prokaryotees

wo classes of transcription terminators, Rho-dependent and Rho-independent, have been identified throughout prokaryotic genomes. These widely distributed sequences are responsible for triggering the end of transcription upon normal completion of gene or operon transcription, mediating early termination of transcripts as a means of regulation such as that observed in transcriptional attenuation, and to ensure the termination of runaway transcriptional complexes that manage to escape earlier terminators by chance, which prevents unnecessary energy expenditure for the cell. Rho-dependent terminators[edit] Rho-dependent transcription terminators require a protein called Rho factor, which exhibits RNA helicase activity, to disrupt the mRNA-DNA-RNA polymerase transcriptional complex. Rho-dependent terminators are found in bacteria and phage. The Rho-dependent terminator occurs downstream of translational stop codons and consists of an unstructured, cytosine-rich sequence on the mRNA known as a Rho utilization site (rut) for which a consensus sequence has not been identified, and a downstream transcription stop point (tsp). The rut serves as a mRNA loading site and as an activator for Rho; activation enables Rho to efficiently hydrolyze ATP and translocate down the mRNA while it maintains contact with the rut site. Rho is able to catch up with the RNA polymerase, which is stalled at the downstream tsp sites.[1] Contact between Rho and the RNA polymerase complex stimulates dissociation of the transcriptional complex through a mechanism involving allosteric effects of Rho on RNA polymerase.[2][3] Rho-independent terminators[edit] Intrinsic transcription terminators or Rho-independent terminators require the formation of a self-annealing hairpin structure on the elongating transcript, which results in the disruption of the mRNA-DNA-RNA polymerase ternary complex. The terminator sequence in DNA contains a 20 basepair GC-rich region of dyad symmetry followed by a short poly-T tract or "T stretch" which is transcribed to form the terminating hairpin and a 7-9 nucleotide "U tract" respectively. The mechanism of termination is hypothesized to occur through a combination of direct promotion of dissociation through allosteric effects of hairpin binding interactions with the RNA polymerase and "competitive kinetics". The hairpin formation causes RNA polymerase stalling and destabilization, leading to a greater likelihood that dissociation of the complex will occur at that location due to an increased time spent paused at that site and reduced stability of the complex.[4][5] Additionally, the elongation protein factor NusA interacts with the RNA polymerase and the hairpin structure to stimulate transcriptional termination.[