no.a77e May 13, 1961 Biology, California Instituto of Technology, Pasadena, during June 1960. We would like to thank Profs. Delbriick for thoir kind support. G. W Beadle and hospitality and finar dit, 2 Co) Be ud, Gor Mol" ; *nelporhy, A. %.,fntern, Symp Ore af Le, 104 abi foam ft Leadon of Miao Se vA deep Bs Aim, Ina, Potiowr, 7B, 987 (1967) Goben, 8 Bac. He. 13,1 (LONG) "Pardee, A B.v and Willa, Tiina. nat Pasteur, #4; 147 883). NATURE 581 ‘Koper, A. immacman, 8,3. Korabere. 8. Mand Some, 3, “ire B.S. Net deal Se Sage TRIS Hales 24 See Ze fits sin Cohen, 88: Xen Chen, Baa Lc “cohen, 8 Bd. Bia Chem’ 274, 28 (943). angen, Leh, J Bacleriol.,"68, 703 (1963). " “Voli nd Astana, a, Yio, 2,149,950) Atendan, and Vola Hy Biochim: Bunnie: Sa, Bb 4 (1088) Dor and Spleesiman, 8 Mel. 306 “Maglio M StL. Wand Vinograd, J, Pro. U.S, at, dead. Sheds Nal bk a Jagifaiaon, J.D, Sebealoger,D., and Hollingworth, tah Bit, 1 221 (1980). na E-obete, He Band Britten, R. J, Poe, 0.8. Nat ‘a Tas) Gros Hai Ober, W, Katana CG, Rlsebrough, RW. nd Watson J’be en folowiag a treme Said ss id rote, “Rtn nd ati Sl, ae, Tmt Sata al ee Pe dead saa tah “ Vali Astachan, Ta and Counteyman, J. Ya, Filo, 8, 8 (1958). zi * Yeas, M., and Vincent, W. 8, Proc. U.S. Net. dew Se, 4, 904 ‘a0, ' UNSTABLE RIBONUCLEIC ACID REVEALED BY PULSE LABELLING OF ESCHERICHIA COLI By Drs. FRANCOIS GROS and H. HIATT ‘The Institut Pasteur, Paris Dr. WALTER GILBERT Departments of Physics, Harvard University ‘AND Dx. C. G. KURLAND, R. W. RISEBROUGH and Dr. J. D. WATSON ‘The Biological Laboratories, Harvard University HEN Escherichia coli colls aro infocted with 7 oven bacteriophage particles, synthosis of host proteins stops!, and much if not all now protein synthesis is phage specifict. ‘This aystom thus pro- vides an ideal modal for obsorving the synthesis of now proteins following tho introduction of specific DNA. Inparticular, wo should expect the appearance ‘of phage-specific RNA, since it is gonorally assumed that DNA is not a ditoct template for protein syn- thesis but that its genotic information is transmitted to a specific soquenco of bases in RNA. Tt was thus considered paradoxical when it was first noticod? that, following infection by the 1? oven phages, net RNA. synthosis stops even though protein synthosis con- tinuos at tho rate of the uninfected bacterium, This could mean that DNA sometimes serves as a direct template for protein synthosis. Alternatively, net RNA synthesis may not be necossary so long as there exists the synthesis of « gonstically specific RNA. that tums over rapidly. This possibility was first suggested by exporimonta of Horshoy*, who, in 1053, reported that 7? infected colls contain a metabolically active RNA fraction comprising about 1 por cont of the total RNA. Several years later, Volkin, and Astrachan* reported that’ this metabolic RNA possessed baso ratios similar, if not identical (con- sidering uracil formally equivalent to. thymine), to those of the infecting 72, DNA. By 1958 they extended their observation to 77 infected cells, where tho RNA synthesized after phags Infection had bate ration silar to thos ofthe phage t wPN)D During these years, ovidenoe? scoumulated that ‘the sites of much, if not all, protein synthesis are the ribosomal partioles, and it was thought mort Maly that ribosomal RNA ‘was gonotically specie, with each ribosome possessing & base. soquonoe which coded for 8 spooifie amino-acid. sequent fons rbot somo pewein hyphen) “Dit verotion of this hypothesis was lacking, and its proponents? wore troubled. by. tho fact: that, excapt, fer: phage. specific RNA, "it. was impossiblo to find. any correlation within a” given organism botwoon the base ratios of DNA and RNA. “Moreover, tnoco was 40 evidence that phage-specific RNA was ribosomal Nomura, Hall and Spiegelman* have recently Aiscovered) that following 2 infection there. is no synthesis of typical (e00 below) ribosomal RNA and that the phage-spooifie RNA sediments at a slowor rato (86) than ribosomal RNA (I6o and 23), ‘Tho gonetio information for the synthosis of phage. specific proteins does not reside in tho usual ribosomal ENA. Instead, if we asoumo that tho synthosis of phago-specific proteins also occurs on ribosomes, thon the phage.spocifio RNA might be viewed as a ‘messon ger" (to uso the torminology of Monod and Jacob") Which carries the genetic information t0 tho ribo. somes. Furthermore, unless wo postulate that there exist two different mechanisms for protoin synthesis, there should also exist within uninfected normal ells RNA. moleoules physically similar. to. ‘tho ha specific RNA and having base ratios similar’ to its specific DNA. 29582 oa /\ . ft" : fo bag YW f a “ ca ar i 4, Sodlmentatio of pulse abled 20-+ Mgt extract, on EOS, Au cxtrch nip Pig True ae e238 gn LBS ‘on sucrose gradient, Drop collection and radioaed veh nesoure: ‘entawere made as decrbed' hn Ree 1 How wo prosnt ovience that RNA moloules physically similar to phago-speciflo RNA oxist. in normal E. coli cells and that, under suitable ionic conditions, they are associated with ribosomal Particlos. Experimental plan. Most (80-85 per cont) RNA in actively growing Hecherichia coli colls is found in ribosomal particles composed of 64 por cent RNA ‘and 36 per cent protein'', Thoro are two sizos™® of, ibosomal RNA, 16s (molocular woight = 5:5 x 10°) and 23s (molecular weight = 1-1 x 10"). ‘The 162 RNA is derived from both 302 and 50s ribosomes, while 23s RNA is only found in 50s ribosomes, ‘The other principal (10-16 por cent) form of RNA in E. colt is soluble RNA (now moro appropriately called transfer RNA), which functions in the move. mont of activated amino-acids to the ribosomes. At Toast 20 (ono for each amino-acid) different transfor RNA molecules exist", all of which havo molecular 30 NATURE May 13, 1961 vox 100 weights about 25,000 and sodimentation constants of 4a. Collectively, ribosomal and transfer RNA comprise at least 95 per cont of Z. coli RNA. ‘Thus messenger RNA, if present, ean amount to at most only soveral er cent of the total RNA. Now if the mossonger wore stable, only @ corresponding fraction of newly synthesized RNA could be messenger RNA; the great majority of new RNA being the metabolically stable ribosomal and transfer RNA’s. If, however, RNA is tuming over (as is ed by the original Hershey oxperimenta) then # much largor fesction of nowly made RNA must bo = For examplo, if the mossonger funetions only onos for tho synthesis of a singlo protein molecule, ite lifetime might be only soveral seconds. In this event, if we look at the RNA synthosis ocourri during a very short interval, then most of the newly. synthosized RNA would bo’ messenger oven though {hi fraction may comprise only 1 por cont of tho total 0400 i Optical density 260 FM AETER_ ana 10 20 20 ‘Tube namber Hig. 8, | Sedimentation of T2-infeeted 10-4 Mo* extract, An Shichi SO Stet of elfen eh 2 20 and labelled with phosphorus-95 between the minutes after (nection. han ae in Fig. ts eveen’ seaeg weet ‘ented with ribonuclease (10 yf.c) to determine the backgrotand ‘of label natin CA 0150 Optical denaity 260 s g Ye 4, Sedimentation of 72-nfsted 10- fg extract long run, extract ae In Fig 6 for At oe at 86 000 2a Ee: aves feared ts in Pignears May 13, 1961 Wo have thoroforo exposed E. coli cells to short pulses (10-20 soc. at 25° C. where the time of genora- tion is about 90 min.) of radioactive RNA precursors (#P or “C-uracil), rapidly chilled the cells with grashed ico and 14/100, anid, prepared. cell-too extracts by lumina grinding, rxyribo- nuclease at Sy/ml., and examined the newly made RNA using tho” sucroso-gradiont centrifugation technique’. In some oxporiments ™C-uracil was given to colls of @ pyrimidine-roquiring mutant (B148) which was briofly starved (30 min. at 25° C.) for uracil. No difforencs has been soon between the propertios of RNA labelled in those two ways. Starved colle incorporate more radioactivity, and ‘they were used in most of the experiments reported below. Reaults. Radioactive uracil (or phosphorus-82) is incorporated in RNA within sovoral soconds after addition of the isotope. ‘Tho RNA labelled by 10-20 800. pulses is stablo at 4° C. in cell-free extracts where ribonuclease and polynucleotide phosphorylase aro i Optical density 260, 8 Wig. 5, Pulee-ibellod active 70e Hboeomes in 10-1 Me!*, An fxtiac of ely nbelld with a 26-ec- pulse of “C-urad mate ie 30+ 3 Set wae puiteg"by dogo seneifuatons at 4,000 emi’ see a as 8% Loa i Goat Ta Those: itspended pect was run a8 for Pig 0-200 200 Connts/min g Optical density 200 3 10 20 30 ‘Tube number Sedimentation of pukedabeled 10-+Mgt extract in Magnesium Was added to the 10" Mage ea ar rig reo beng to 10's A the eae Ae gaat ast ‘on Sarge gradient in 10 Mtg, bx NOT MW, fore he ‘4simin. ‘Tho measurctents were thade as dseribod fer Wig NATURE 583 ot Sow 4 i i i vod z 8 10 20 30 ‘Tube number Wig. 7, Sedimentation of 12-Ifbeted 10-+ Ma’ extract, An Gutiack in 10°" A Mg of ocis Infected with 72 ant inbetied frit phosphorus-32 from tho aeonnd to the Af alae ettes Tarectfon, "Hun om a sucrose gradient iu 10-" a Agi for 2 he. Bin." at 25,000 cpm.” Drop colletlon and Fadioeetiely ‘moasurements aa for Fig. not active, ‘Traces (Iy/ml.) of ribonuclease degrade it under conditions where RNA. in ribosomes “is untouched. This suggosts that it oxists loss protected than bound ribosomal RNA. Similarly, addition of phosphate permits tho polynucleotide phos- Phorylaso' in the coll extract to degrade proferenti. ally pulse-labolied RNA. Our experiments thus as ‘@ matter of routine avoid tho use of phosphate bulfors. Figs. 1 and 2 show how RNA labelled by a 20-800. ‘exposure of uninfected oolls to MO-uracil sediments in a sucrose gradiont. Tho ooll-froo extract contains 10-4 Mf magnesium ions in which 30+ and 609 ribo somes predominate. ‘Tho majority of the radioacti ity ia not associated with ribosomes but moves with & 14-168 poak. There is also RNA which moves more slowly, in addition to a faster forward fraction moving at 70s. Tho slower sedimenting fraction shows up moro clearly in the longer centrifugation shown in Fig. 2. Horo a sharp 14-16s componont is seen together with material sedimenting at 4-89. Figs. 3 and 4 illustrato experiments with extracts from 72 infected eolls exposod to phosphorus-32 from 2 to 6 min. after infection. ‘They are similar, if not. identical, to Figs. 1 and 2. Both havo a’ major 14-168 component, a slowor trailing fraction, and about 10 por cont of the material moving at 70s.” ‘Tho 708 component can be purifiod by threo 1-hr. cont: fagations ot 40,000 rpm. with 10-* M magnesium, ions to concentrate the faster-moving ribosomes of 1. Fig. 5 shows the purified ribosomes consisting largely of 508 particlos together with a now visible 70s component. ‘The radioactivity, however, sodi- ments as 70s ribosomes. Still more label is attached to 70s particles when extracts aro made in 10-* M magnesium ions or when additional magnesium ions aro added to 10 Mf extracts. Figs. 6 and 7 again illustrate the parallel apposrance of normal and phago-infocted extracts. About 30 por cont of Inbel sedimonta with the 70s and 100s ribosomes, with tho specific activity of 700 ribosomes genorally groater than that of 100s ribosomes. When slightly lowor concentrations of um ions aro used to givo similar amounts of 30s, 506 and 705 ribosomes, label is specifically associated only with 70s particles and no label is in 50s ribosomes. ‘The radioactivity sodimenting about, st