Ooyen, Albert Johannes Joseph van

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   University of GroningenControl of ribosomal RNA synthesis in Escherichia coliOoyen, Albert Johannes Joseph vanIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1976Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Ooyen, A. J. J. V. (1976). Control of ribosomal RNA synthesis in Escherichia coli. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-20236 5ppGppr i.t is' 1c  e f f ec to r'nerase-rrs  in  v ivo intvolved cannotmechanism inr3-3141rsomes (Nomura,tp 733-7 45 ,! ,  J n D . ,  1 9 7 2 ,n.  Biophys.e n ,  P . ,  L 9 7 6 ,,h in.  Biophys.and Gruber,  M. ,Introl ofa a r d ,  N . O . ,  e d s .  ) ,openhagen,i o l .  Chen .  250 ,43 r  72L -775. ,  L 9 7 5 ,n l s t r y  9 ,CHAPTER VIISUMMARY AND CONCLUSIONSIn th is  chapter  we rev iew our  resul ts  and the most  recentI l terature on the subject .  Speci .a l  a t tent ion is  g iven to acompar ison of  in  v j . t ro and in v ivo resul ts  on rRNA synthesis ,for  the u l t imate goal  of  i -n  v i t ro s tudÍes is  to  construct  arRNA synthesiz ing system conta in ing a l l  regulatory e lementsthat  are inrro lved in v ivo.  As wj . l l  be shown below,considerable progress has been made in that  d i rect ion,although a number of questions remain to be answered.The most  obvious precondi t ion for  correct  in  v i t rot ranscr ipt ion 1s in i t j -a t ion and terminat ion of  the RNA chainsat  the r lght  s i - te .  Studies of  Pet t i john (1)  and Jdrgensen andFi i l  (2)  in  d i f ferent  in  v i t ro systems have shown thatt ranscr ipt ion occurs in  the sequence 165-235 which is  thesame as in  v ivo ( l ) .  Fur thermore,  the sedimentat ion constantof the rRNA made i-s approximately 3OS (2) equal to that ofthe rRNA precursoï  in  v ivo (4) .  I t  can be concluded that  rRNAtranscr ipt ion in  v i t ro s tar ts  and ends at  or  near  the in  v j -vosi tes.  The f ina l  answer to th ls  must  come f rom sequencestudles of  the in  v i t ro product  and of  the in  v Ívo precursorcOne of  the character is t ic  features of  in  v ivo rRNAsynthesis  is  the h igh in i t ia t lon f requency of  the RI . {Apolymerase molecules on the rRM cis t rons which is  5O-1O0t i rnes that  of  the average mRNA cis t ron t ranscr ibed (5) .  As aresul t ,  up to 6A% of  a l l  RNA made in fast  growing E.  co l icel1s is rRI.IA (see below) although less tlnan L% of the totatDNA codes for rRNA. In purif ied systems only 2-1O% rRNAsynthesis  is  found (1,  6-9) .  The d i f ference between the invj-vo situation and the ptrrif ied systems may be due to a lowrRNA synthesis  in  v i t ro,  for  instance through loss of  factorsposi t ive ly  in f lueneing rRNA synthesis  on one hand or  to anincreased non-rRl t iA synthesis  in  v i t ro on the other .We have studied th is  problem by us ing so-cal Ied nucleoids,the folded chromosomes, prepared by methods original-lydeveloped by Pet t i john et  aIo (1O).  These nucleoids werethought to be a more physiological template than DNA preparedby convent ional  procedures involv ing phenol  ext ract ion:  theDNA in the nucleoids contains less breaks which may serveas ar t i f ic ia l  s tar t ing points for  RNA potymerase (11)  and has6 6a ter t iary s t ructure resembl ing the in t racel lu l -ar  s t ructure(L2> "  The nucleoids could only  be prepared in lowconcentrat ions which is  an exper imenta l  d isadvantage,In Chapter  IV (see a lso ref .  9)  is  shown that  57+4% ofthe RNA synthesized by RNA polymerase present  in  nucleoidsfrom fast  growing cel ls  couLd be competed by rRNA f romr ibosomes .  As  expec ted ,  t h i s  i s  c l ose  to  t he  i n  v i vo  va lue .Pato and Von Meyenburg (13)  found that  in  fast  growing ce11s63% of  a l l  act ive RNA polymerase molecules is  engaged instable-RNA synthesi -s .  This  value was later  corrected to 57%(see ref , t4). If we assume that 90% of the stable RI,[A 1srRNA (14)  than we arr ive at  5L% rRNA which is  c lose to thevalue we found.  I t  s t rould be noted that  about  20% of  the inv ivo rRNA precursor  molecule is  not  conserved in v ivo,i nd i ca t i ng  t ha t  i n  f as t -g row ing  ce1 l s  abou t  60% o f  a l l  ac t i veRNA polymerase molecules is  engaged in rRNA precursorsyn thes i . s .I f  RNA polymerase is  added to the nucleoids i t  synthesizesrRNA at about the same rate as the endogenous enzyme but non-rRNA at a much higher rate resultlng in a low percentage rRNA(3-5%) as demonstrated in  Chapter  IV"  I t  is  in terest lng thatGiorno et  aI .  (15)  us ing nucleoids f rom s lowty growing cel lsobserved that  the added enzyme synthesizes more rRNA thanthe endogenous one.  Thus no specia l  factor  f rom fast-growingce l l s  seems  to  be  respons ib le  f o r  t he  h lgh  i n l t i a t i on  ra teon the rRNA cistrons cIn Chapter  IV ev idence has been presented that  thein l t ia t ion rate on the r ibosomal  c is t rons is  maximal  in  oursystems;  i t  is  determined by the c losely packed RNA polymerasemo lecu les ,  i oe .  t he  e longa t l on  ra te .  S ince  the  e longa t i onra te  i s  on l y  1O-2O% o f  t he  i n  v i vo  va1ue ,  t he  i n l t i a t i on  ra teof  the RNA polymerase molecules on the rRNA c ls t rons in  v i t ro1s only LO-2O% of  the in  v ivo value in  fast  growing ce11s.We expect  that  an increased e longat ion rate 1n v i t ro wi l llead to an increased in i t ia t ion f requency unless factorsmiss lng in  pur i f ied systems are necessary for  h i .gh rRNAln i t i a t  i on .From our work wi th the nucleoids,  we conclude that  thepercentage rRNA made in vitro 1s probably lower than the lnv ivo maxj-mum for  2 reasons.  F l rs t ly ,  a  decreased in i t ia t ionrate on the rRNA c is t rons due to a decreased e longat lon rate.Secondly,  non-rRNA synthesis  1s much more abundant  in  v i t rothan in v ivo under condj . t lons of  maximal  growth"lu1ar  s t ructuren Lowdvantage.that 57+4% oft  in  nucleoidsrRNA fromin v ivo value.st  growing cel lss engaged inorrected to 57%stable RI.IA iss  c l ose  to  t het  20% of  the 1nd i -n v ivo,60% of  a l l  act lveprecursords i . t  synthesizess enzyme but non-tl percentage rRNAinterest ing thatly  growlng cel lsore rRNA thanrom fast-growingnl t la t ion ratethat  themaximal in ourked RNA polymerasehe elongatione  i n l t i a t i on  ra te3i .s t rons in  v i t rogrowing cel ls"i n  v i t r o  w111Iess  f ac to rsr high rRNAlude that theiler than the inl sed  l n i t l a t i onelongat ion rate.rndant 1n vltrovth "67Considerable evidence has accumulated that ppGpp isinvolved in the regulatj-on of rRl,[A synthesis in vivo in E.co l i  and  o the r  p roca ryo tes  (16 ,  see  re f .  17 ) .  I n  ea r l i e r  i nv i t ro s tudles no specl f ic  inh ib i t ion of  rRNA synthesis  byppcpp could be found (617r18) .  As pointed out  in  Chapter  Vth j -s  was probably par t ly  due to the use of  rather  insensi t ivehybr id izat ion methods and par t ly  to  the use of  unfavorablesal t  concentrat i .ons.  Wi th our  h ighly  sensi t ive hybr id izat íonsystem descr ibed in deta11 1n Chapter  I I I  we could show thatppcpp speci f ica l ly  inh i -b i ts  rRNA synthesis  in  pur i f ied invitro systems containj-ng DNA and Rl.lA polymerase as the onlymacromolecular  components (19r2O, see a lso Chapters V and VI) .This f ind j -ng was later  extended to crude systems (Chapter  V) .The most important f indings concernj.ng the specific inhibit ionof rRNA synthesis by ppGpp are:(i) It is independ.ent of the genome which the rRM cj-stronsform par t  of .  l t re  found the ef fect  wi th E.  co l i  and, td5i lv  DNA.( i i )  I t  was found wi th three d i f ferent  RI , [A polymerasepreparat ions prepared in three d i f ferent  laborator ies.( j - i i )  I t  is  speci f ic  for  ppGpp.  Only pppcpp mimics theef fect  of  ppGpp.(fv) The Ki for the inhibit lon of rRM synthesis by ppGppi s  0 . 1 5  n M .(v)  The inhib i t ion is  exer ted at  the leve1 of  in i t ia t ion.( i )  and ( i i )  show that  the ppGpp ef fect  is  not  a specia lfeatur:e of a certain DNA and RNA polymerase preparation. Ourfinding in purif ied systems now has been confirmed by otherl n v e s t i g a t o r s  ( 2 r Z L ) .( i i i )  suggests that  ppGpp l tse l f  and not  a metabol i te  ofppcpp is the effector of rRM synthesis in vivo, and showsthat ppGpp does not act via substrate homology with GTP.(1v)  and (v)  are in  agreement  wi th the f ind ings onregulation of rRl{A synthesis in vivo during amino acidstarvation where it has been found that the apparent K, forstable rRI{A asqunulation is 0.15 mM ppGpp (22) and that theinhi -b i t ion is  exer ted at  the level  of  rRNA chain in i t ia t ion(23)  "A compar ison of  in  v l t ro and in v ivo resul ts  s t ronglysuggests that during amino acid starvation ppcpp alone isresponsib le for  the sharp decrease in rRM synthesis .  I texer ts  i ts  in f luence by inhib i t ing rRNA chain in i t ia t ion bydl rect  in teract j .on wi th the RNA polymerase-r ibosomal  DMsys temc68The regulation of rRNA synthesis during exponentJ-al growthand shi f ts  in  the growth rate is  1ess wel l  understood.  FÍ i let al. (22) }:.ave polnted out that the variation in ppcppconcentrat ion as a funct ion of  exponent ia l  growth rate i .smuch too smal l  to  suf f ice as the sole contro l l ing e lementfor  the wide range in s table RNA accumulat ion observed.  Inamino acj .d s tarvat ion much larger  var iat ion in  ppcppconcentrat lon is  corre lated wl th equal  changes in s table RMaccumulat ion.  A s imi lar  conclus ion was drawn f rom exper imentsper formed dur j .ng shi f ts  in  growth rate (24r25) .  Therefore,an addl t ionar  contror  system for  rRNA accumulat ion besides thecontro l  by ppGpp was pï .oposed (22) .  Several  possib i l i t iesex i s t  f o r  such  an  add i t i ona l  con t ro l  such  as ;  ava i l ab i l i t yof  RNA polymerase molecules (26) ,  a posi t ive ef fector  of  rRMsynthesis  (22124-26) ,  rRNA breakdown (27) ,  the phantom spot(28) ,  or  a factor  in f luencing the in teract ion between ppcppand RNA polymerase.In v i t ro s tudles have only been concerned wi th posi t iveef fectors of  rRNA synthesis .  The exis tence of  a posi t ivecontro l  factor  has been proposed by several  invest igatorsnTravers (Z)  suggested the e longat ion factor  EF-Tu as aposi t ive ef fector  of  rRNA synthesis .  Unfor tunate ly ,  at temptsin several  laborator ies to reproduce h j -s  resul ts  have fa i led(216120 ,  see  a l so  Chap te r  V ) .  I n  a  recen t  s tudy ,  B lock  (29 )c la ims the ex ls tence of  a factor  in  a crude system posi t ivetyj -nf luencing rRNA synthesis .  This  factor  however,  has notbeen pur i f ied;  h is  resul ts  may a lso be expla ined by changesj -n the RNA polymerase to DNA rat io  in  the react ion mixture.Thus the ex j -s tence of  a posi t ive ef fector  of  rRNA synthesisis  far  f rom establ ished.  I t  is  quest ionable whether  such afactor  is  needed" Udvardy and co-workers (3Or31) found thatthe lnteraction between RNA polymerase and the rRNA promotersis  much st ronger than the in teract ion between Rl . [A polymeraseand the average non-rRNA promoters.The in v i t ro s tudies lead to the conclus ion that  twoprocesses are par t  of  the regulat ion of  rRNA synthesis  inv ivo.  These are the st rong interact ion between RNA polymeraseand the rRNA promoters whj .ch a l lows a h igh in i t ia t ion rateon the rRNA cistrons both in vivo and in vitro and ppGppwhich decreases the af f ln i - ty  of  RNA polynerase for  thesepromoters.

Control of ribosomal RNA synthesis in Escherichia coli

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Control of ribosomal RNA synthesis in Escherichia coli

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