Isolation, purification and properties of 5 s ribosomal RNA: a new species of cellular RNA

J. Mol. Biol. (1967) 23, 441-458

Isolation, Purification and Properties o f 5 s Ribosomal R N A : a N e w Species of Cellular R N A DO1,TALD G. COM_BAND TOVA ZEIL~vI-WILLNER

Department of Biological Chemistry, Harvard .Medical School, Boston Massachusetts, U.S.A. (Received 5 September 1966, and i7~revised form 26 October 1966) The isolation procedure for a new species of ribosomal I%~A is described. The name 5 s ribosomal I~NA is proposed for this component. I~ has been found to occur in ribosome preparations from many different organisms. Approximately one molecule of 5 s rI~NA is associated with the ribosome. It has a chain length of 120 in ribosomes from Eschcrichia cell and 148 in ribosomes from Blas~ocladiella emcrsonii. The major base composition is similar to transfer RNA but no methylatcd bases can be detected. V~rohave estimated that about 0.5 residue of pseudouridine occurs per molecule. Other physical and chemical properties are described. 1. I n t r o d u c t i o n An RNA species resembling tRNAJ" in major base composition but without amino acid-accepting activity or methylatcd bases was first described by Rosset, hlonier & Julien (1964) in Escherichia cell and, independently, by Comb & Katz, (1964) in the aquatic fungus Blastocladiella emersonil. The former investigators demonstrated that this species of RNA, called 5 s I~N'A, was attached to the 50 s ribosome subunlt and that the chain length was about 105 nueleotides. Studies from this laboratory have confirmed the fact t h a t this unique I'r component is present only on ribosomes. Isotope experiments indicated that label catered this RNA species (called transfer-like ~ N A in publications from this laboratory) in the nueleolus prior to tRNA, and in vitro studies suggested that it was a better methyl-accepter than tI~NA (Comb & ]Katz, 1964: Comb, Sarkar, DeVaUet & I)inzino, 1965). ]~rom these observations we proposed that transfer-like I~BTA may be a precursor of tRNA. This hypothesis proved to be wrong, however, since it was subsequently demonstrated (Zehavi.Willner & Comb, 1966; Hayward, Legault-Dcmare & Weiss, 1966) that transfer-like RNA (5 s rRNA) did not contain base sequence homologies to tRNA. Furthermore, the methyl-accepting activity previously ascribed to this I'r species has been separated from it and demonstrated to be due to submethylated tI~NA attached to ribosomes (Sarkar & Comb, 1966). This submethylated tRNA has chromatographic properties on methylated albumin column~ very similar to those of 5 s rRNA. Since it appears that 5 s rrCNA is a distinct species of ribosomal RNA, we shall hereafter adopt the name 5 s rI~NA to denote tlfis new species of cellular RBTA. I t appears that 5 s rl'CNA is ubiquitous, since it has been reported to be in yeast ribosomes (BIarcot-Queiroz, Julien, Rossct & 5Ionier, 1965) liver ribosomes (Galibert, I" Abbreviations used: tI~N'A, transfer P,NA; rR~A, ribosomal I ~ A : ~, pseudouridlno. 441

442

D. G. COI~IB A N D T. Z E t I A V I - W I L L I N ' E R

Larsen, I,clong & Boiron, 1965), sea urelfin r i b o s o m e s (Comb, K a t z , B r a n d a & Pinzirto, 1965), a m p h i b i a n ribosomes (Bro~)~n & L i t t n a , 1966) a n d r e t i c u l o c y t e ribosomes ( Z c h a v i . W i l l n e r & Comb, u n p u b l i s h e d results). Sehleich & Goldstcin (1966) h a v e s e p a r a t e d w h a t a p p e a r s to be 5 s r R N A from p r e p a r a t i o n s of/!7, coli t R N A b y S e p h a d e x gel f i l t r a t i o n a n d r e p o r t e d t h a t i t has a chain l e n g t h o f a p p r o x i m a t e l y 122 mlcleotidcs. T h e p r e s e n t p a p e r describes a m e t h o d for t h e isolation o f 5 s r R N A from b o t h E . coli a n d B. emersoni~ wlfich yields a n e a r l y homogeneous p r o d u c t . P h y s i c a l a n d chemical p r o p e r t i e s o f 5 s r R N A , w h i c h m a y p r o v i d e some insight as to t h e s t r u c t u r e o f this molecule a n d i t s biological f u n c t i o n on t h e ribosome, will also be described. A s u b s e q u e n t p a p e r f r o m this l a b o r a t o r y will d e m o n s t r a t e t h a t 5 s r R N A b i n d s to two sites on d i s s o c i a t e d ribosomes: one o f these is specific for 5 s r R N A , whereas b i n d i n g to t h e o t h e r site can be d i s p l a c c d b y tRIffA.

2. Materials and Methods (a) Growth of B. emersonii The unicellular aquatic fungtts, B. emersonii (Phyeomyeetes) was grown in the liquid culture medium described b y Cantlno & Horenstein (1956). The medium consists of the following, in 1.0 liter: 1.25 g of Peptone (Difeo), 1-25 g of yeast extract (Difeo) and 2 g of dextrose. Solid medium is prepared b y the addition of 2 % agar. The yeast extract employed in most of this work contained 0.370 m-mole inorganic phosphate a n d 0.099 m-mole organic phosphate per g. The Peptone contained 0.002 m-mole inorganic phosphate and 0.068 m-mole organic phosphate per gram. When isotopes were employed in the meditun a n d the a m o u n t of either Peptone or yeast extract was reduced, inorganic phosphate was added to bring the total phosphate content to the above level. 2~Iotilo spores were harvested from a "lawn" o f B . emersonii growing on an agar medium (200 ml. in a l-liter R o u x bottle) b y the addition of 10 to 20 ml. of distilled water when the plants are r e a d y to dlseharge motile spores. BIotilo spores (7.5 to 10 • 107) were inoculated into 1 liter of liquid medium in a 2-1. Erlenmeyer flask and the culture was incubated a t 25~ with aeration b y means of a r o t a t o r y shaker. Cells were harvested after 5 to 6 hr, when t h e y are still in their period of rapid growth, and stored a t -- 20~ until used. :For measuring spore density 1.0 Ae0o unit in the Zeiss speetrophotometer is equivalent to a p p r o x i m a t e l y 0.5 • l0 s motile spores/ml. F o r a more complete description of the life cycle o f B . emersonil and other variations on tim lifo cycle, the reader is referred to a r e c e n t review b y Cantino & L o v e t t (1964). (b) Growth of E. cell B The ceils were grown at~ 37~ with aeration in a medium containing the following in 1.0 liter: 2 g :NH4CI, 1.0 g NaCl, 0.01 g MgSO4,7I-I20 , 6.0 g :Na2IIP04, 3.0 g KHaPO4, 5.0 g Casamino acids (Difco) a n d 20 g dextrose (autoclaved separately). The cells were harvested during exponential growth and stored a t --2O~

(e) Isotope Carrier-free s2P l was obtained from the Cambridge :Nuclear Corporation, Cambridge, ~Iass.; [14C]uraeil and L-[14C-methyl]metllionino were products of the /flow England Nuclear Corporation, Boston, ZIass.; a n d [x4C-earbonyl]isonieotlnie acid hydrazide was purchased from the :Nuclear Chicago Corporation. The above isotopes were used without dilution of specific a c t i v i t y except the [x4C:earbonyl]isonicotinie acid hydrazlde, which was diluted to a specific activity of 2.16 t~c/t~molo with unlabeled compound obtained from the E a s t m a n K o d a k Company. (d) .~Iethylated albumS~ column chromatography l~Iethylated albumin was prepared from erystaUized bovine albumin (Mann l~eseareh Laboratories, Inc., :Now York, xN.Y.) as described b y Mandell & Hershey (1960). The

5 s ~IBOSO.MAL I~l~A

443

mixture of mcthylatcd albumin and IIyfio-supcrcol was also that described b y the above authors. Columns of varying size were employed, depending on the type and a m o u n t of I~NA to be chromatographcd. Good separation of 5 s rRNA from t R N A was obtained when 50 A26o units of 5 s rl~NA or less were chromatographcd on a column 2 cm • 36 cm with a linear salt gradient of 750 ml. each of 0.3 .~I-NaC1, 0.02 .~[-Tris (pII 7.3) and 1.5 ~i-NaCl, 0.02 .~I-Tris (pII 7.3). ~Vhcn the column size was changed, the gradient volume was adjusted to m a i n t a i n the same ratio of gradient volume to column volume as above. The columns were used once a t room temperature and discarded. (el DEAE-celluloso chromatography Chromatography of a mixture of 5 s rRNA a n d t R N A on DEAE-colluloso a t room temperature does not separate these two species. IIowover, chromatography at 80~ (Goldthwait & Kerr, 1962), where only the r a n d o m coil forms exist, separates these two components, with 5 s rRNA cluting at a higher salt concentration than t R N A due to its longer chain length. The following conditions were used. The D E A E C1- form cellulose column (I cm • 10 cm) was maintained at 80~ with a jacketed column and circulating water bath. The column was first washed at this temperature with 1.5 .~x-NaCl, 0.02 ~-Tris (plOt 7.3), followed by 0"3 ~z-NaCl, 0.02 ~t-Tris (pit 7-3). The sample was applied (10 to 20 A260 units) and eluted with a linear gradient of 150 ml. each of 0.3 ~z-NaCl, 0.02 ~z-Tris (pit 7.3), and 1.5 ~-NaCl, 0.02 xt-Tris (pit 7"3). The gradicnt solutions were maintained a t room temperature. (f) 200-transfer countercurren~ dlstributio~ The procedure of Apgar, IIolloy & l~Ierrfll (1962) was followed with a n ]=I. O. Post 200tube fractionator. The sample, 0-8 A2Go u n i t of 5 s [32P]rtXNA (800,000 cts]min) was dissolved in the first tube. After the run, 1.0-ml. portions of the upper phase were counted in Bray's scintillation mixture (Bray, 1960) and total counts per tube calculated from partition coefficients. (g) Ghain-length determination The method of l~Iidgloy (1965) was used. This involves oxidizing the 3' terminus with periodate and treating the aldehyde formed with [x4C-carbonyl]isonicotinie acid hydrazide. The procedure was modified in t h a t after formation of the hydrazone the R N A was precipitatcd with 2 vol. of ethanol (pII 4.8) and a drop of saturated NaCl. The lqNA precipitate was washed twice with 70% ethanol ( p i t 4.8) to remove most of the unreacted radioactive material. The I~NA was then chromatographed on a methylated albumin column at p i t 6.0 (0.01 ~I-phosphate buffer (pit 6.0) was substituted for the Trls buffer in the standard procedure). The specific activity (cts/min/A26o unit) of each tube throughout the absorbaney profile was determined and the specific activity (cts/min]~molo) of the [14G]isonicotinie acid hydrazide was determined under identical counting conditions as the [14G]RNA. (h) Preparation oJ 14G.labeled 5 S r R N A B. emersenii was grown in 1 liter of medium modified to contain only 0.125 g of yeast extractfl, and 50 #o of [14C]uracil (30 po/~molo). After 5 hr of growth, the cells were harvested and 5 s rl~NA isolated as described under Results. (i) Methylated base content of 5,7 rl~NA The methylated base content was dctcrmincd b y ~ vivo labeling with r,-[14C-methyl] methionine. B. emersonii was grown for 5 hr in medium modified to contain only 0.125 g of Poptono]l. A t this time, 50 ~c of [14C-methyl]methionino (13-1 ~c/#molo) was added. After 30 m i n of additional growth, the cells were harvested. The t R N A and 5 s rl=tNA were isolated and chromatographcd on methylated albumin columns for comparison of radioactivity profiles. E. colt B was grown for 4 hr on the medium described above b u t modified to contain only 0-I g of Casamino acids]l. At this time, I00 ~o of L.[~C-n~thyl]methionino was added to 1 liter of cells and after 10 m i n of additional growth the cells were rapidly cooled to 0OG, harvested, and t R N A and 5 s rRNA isolated.

441

D. G. CO~IB AND T. Z E H A V I - W I L L I ~ E R

(j) 13ase analysis The method of Is & Comb (1963) was used for RNA isolated from methylated albumin column peaks. To determine the pseudouridlno content of 5 s rRNA, the eellg were labeled in rive with [2-a4C]uraeil. After isolation and rechromatography of the 5 s rRNA peak on methylated albumin columns, the [14C]RNA was hydrolyzed with alkali. The Up peak, isolated according to I~atz & Comb (1963), was treated with alkaline phosplmtaso and the nucleosides isolated after deionizatlon with mixed-bed resin. The [x4C]nueleosides were ehromatographed with carrier pseudouridino for 48 hr by descending paper chromatography in water-saturated butanol. The pseudouridine (R a 0.5) and uridino ultraviolet-absorbing spots were marked and the paper cut into strips for counting in a toluene scintillation mixture. (k) PrcparaHon of 5 Yl [aeP]rRNA. 13. emersoni~ was grown in the standard medium containing 5 mc of 32Plfl. After 6 hr the cells were harvested and the 5 s rRNA isolated as described under Results. The final yield of highly purified 5 s [32P]rRN'A from 1 1. of cells (3 g we~ weight) was about 0.5 rag, with a specific activity (after 7 days decay) of 2.8 • I08 ets/mln/mg. (I) Other ~nethods Sedimentation coefficients of tlRNA and 5 s rRNA from E. coli were determined with the Spinco model E analytical ultracentrifi2ge. Pictures were taken at 32-rain intervals with the ultraviolet optics. Absorbancy measurements were made with a Zeiss spectrephotometer. The melting profdes of RNA were determined according to the method of BIarmttr & Dory (1962) in 0.15 ~-NaC1, 0"015 ~-sodium citrate (pit 7). For chain-length determinations and other calculations we have used the following relationships: one absorbaney unit (A280) of either tRNA or 5 s rRNA in 0.1 x~-l~laCl (p]~ 7) is equivalent to 125 m/zmole of RNA nucleotide, and 1 mg of the sodium salt of these two R.NA species has a n A 2 s 0 Of 23"2.

3. R e s u l t s (a) Procedure for the isolation of 5 8 r R N A (i) 5 ~ r R N A from E. cell B The cells are suspended in three to four volumes of standard buffer (0.05 ~r-KCI, 0.01 ~t-Tris (pI-I 7"3)) containing 10 -2 ~r-BIgCla and disrupted by sonieation for two to four minutes (Branson sonifier, Branson Instruments, Inc.). Unbroken cells and cell wails are removed by centrifugation for 20 minutes at 30,000 g. Sodium deox-ychelate (5%) is added slowly to the supernatant solution to a final concentration of 0.2% and any precipitate that forms is removed by low-speed eentrifugation. The supernatant solution is centrifuged at 105,000 g. for two hours. The ribosome pellet and sides of the tube are carefully washed once with buffer. Tile. pellet is resuspended in standard buffer containing 10 -2 ~I-1~[g2+ and 0.1% sodium deoxycholate and the ribosomes washed once by centrifugation at 105,000 g for two hours. The pellet is resuspendcd in standard buffer containing 10 -4 ~I.Mg 2+ and dialyzed overnight ag 0~ against an excess of the same buffer to ensure that the ]~Ig2 + concentration in the ribosome suspension is lowered to 10 -4 ~r. After dialysis, any precipitate that forms is removed by low.speed eentrifugation and the dissociated ribosomes collected by eentrifugation at 105,000 g for four hours: The pellet and sides of the tube are washed carefully with standard buffer containing 10 -4 ~r-Mg2+, and the pellet dissolved in 10 to 20 volumes of 0.1 ~I-lgaC1, 1.0% sodium lauryl sulfate. The solution is deproteinJzed by shaking with art equal volume of water-saturated phenol for 15 mhmtes. The R N A is recovered from the aqueous phase after centrifugation by the addition

5

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rClBOSOMAL I~NA

(o)

445

DNA

0-8 5s

16S

06

04

oE 0-2

o

(b)

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0.6

0"4

0-2

20

40 Fraction no.

60 (8ml.)

80

IO0

Fro. 1. ~Iethylated albumin column ehromatograras of nucleic acids soluble in I ~z-~aCl extracted from E. coil washed ribosomes. (a) Before dialysis against 10 -4 ~.Mg 2 * and eentrifugation to remove tRN'A and D ~ A . (b) After dialysis againsb 10 -~ .~I-SIg2§ and recovery of subunits by ecntrit'ugatlon. The column size was 2 cm X 36 cm. Other details are doscribed under Materials and 3Iothods. o f approximately 0.1 volume of saturated NaCI and 2 volumes o f ethanol. After standing for several hours at - 2 0 ~ the precipitate is collected b y eentrffugation a n d dissolved in a m i n i m u m volume of 0.1 ~I-NaCl (about 200 to 400 A2G0 units per ml.). A n y insoluble material is r e m o v e d b y centrifugation. A t this stage, the R N A solution contains mainly 5, 16 a n d 23 s r R N A . Since only a b o u t 3 % of the total is 5 s r R N A , better resolution on m e t h y l a t e d albumin column c h r o m a t o g r a p h y is obtained if m o s t of the 16 and 23 s r R N A is removed. This is accomplished b y the addition of 4 .~[-NaC1 to a final concentration o f 1.0 ~. The solution is allowed to stand for at least 12 hours a t 0~ for m a x i m u m precipitation of 16 and 23 s rl-CNA; the more concentrated the solution the more complete is the precipitation of high molecular weight R N A . 5 s r R N A is completely soluble in either 1.0 or 2.0 ~I.NaCl. W e have observed t h a t some precipitation of 5 s r R N A m a y occur in 2 :~-LiC1 and therefore feel t h a t NaC1 is the salt o f choice, even t h o u g h 16 and 23 s r R N A precipi. 30

446

D. G. COMB AND T. Z E H A V I - W I L L N E R

tation in 2 ~[-LiC1 is almost quantitative. After centrifugation, the 1.0 xuNaCl pellet is washed with several volumes of ice-cold 1.0 ~-NaC1. The supernatant solutions are combined and the RI~A recovered b y precipitation with 2 volumes of ethanol. The precipitate is dissolved in 0.2 .~[-NaCl, 0.02 ~I-Tris (pit 7.3) and chromatographed on a methylated albumin column. To determine the size of the column required (see hIaterials and ~Iethods), we have assumed t h a t about one-half of the A26o units present in the solution are 5 s rRhTA. :Figure l(b) represents a typical chromatogram of the elution profile of the RNA at this stage of the purification. 5 s rRN'A is eluted as a single symmetrical peak well separated from residual 16 and 23 s rRNA. :For comparison, :Fig. l(a) shows the clution profile of RI~A extracted from ribosomes washed only in 10 -2 ~r-~Ig 2+ (without dialysis against 10:4 ~r-~lg 2+ and ccntrifugation). Under these conditions, the 5 s r R N A peak is badly contaminated with tRNA. Also note t h a t DNA fragments arc attached to these ribosomes and are not removed b y washing when the l~lg2+ is 10 -2 ~r. I

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70

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Fraction no.

Fla. 2. Methylatcd albumin column chromatogram of RNA (and DNA) soluble in 1-0 ~r-NaCl, extracted from B. emersonii ribosomes after dialysis against 10 -~ ~t-Mg2+. The column size was 2 cm X 36 cm. (ii) 5 S r R N A from B. emersonii B. emersonii cells are broken by homogenization with title glass beads (Comb, Brown & Katz, 1963) in standard buffer containing 10 -2 ~r-hlg 2+. The nuclei, nuclcoli, cell walls and glass beads are removed b y low-speed centrifugation. The supernatant solution is centrihlgcd at 30,000g for 20 minutes to remove mitochondria. Sodium dcoxycholate is added to the 30,000 g supernatant solution as above and the 5 s rRNA isolated as described for E. col/. :Figure 2 shows the methylated albumin column chromatogTams of RNA extracted from B. emersonii ribosomes after purification by the above 9procedure.

5 S RIBOSOMAL

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Fro. 3. ~Iethylated albumin column chromatograms of tIChIA and 5 s rRI~A isolated from various cytoplasmic fractions of B. ernersonii after the cells had been given a 30-min pulse of [14C-metTlyl]mcthionlno. Prior to chromatography the fractions were incubated at 37~ (ptI 9) for 30 min to remove traces of aminoacyl I~N'A. (a) The I'r present in the 105,000 g supernatan~ solution. (b) The RKA released from ribosomes by dialysis against 10-a ~-BIg2§ (c) The RNA (and DNA), soluble in 1.0 ~-l~aC1, extracted from the 10-4 ~I.3ig2+ ribosome pellet. The radioactivity profile shows the distribution of I'r containing methylatcd bases. The column size was 1 cm • 26 cm; ( - - O - - 0 - - ) A260;( - - 0 - - O - - ) radioactivity. Figure 3 (absorbaney profile only) represents the total t R N A and 5 s rRNA present in the various cytoplasmic fractions of B . emersonii. Figure 3(a) shows the total RNA present in the 105,000 g supernatant solution and :Fig. 3(b) represents the total RNA released from the ribosomes by dialysis against 10 -4 ~I-Mg2§ After the ribosomes were removed by centrifugation at 105,000 g for four hours, the supcrnatant solution was passed directly over a methylated albumin column. The chromatogram in Fig. 3(e) represents the total R N A (and DNA), soluble in 1"0 ~-NaCl, that was extracted from the 10-4 ~i.hig2 + ribosome pellet in this same experiment. These results demonstrate that 5 s rI~NA is not present in the soluble portion of the cell (trace amounts m a y possibly be present as indicated by the shoulder on the trailing edge of the tRb~A peak). I t is also apparent that little 5 s r R N A is released from the ribosomes when the Big2+ concentration is lowered to 10-4 ~I, whereas t R N A is essentially completely released. We have estimated that about 2.0% of the total RNA A20o units of

44S

D . G . COMB AND T. Z E t t A V I . W I L L N E R

B. emersonii ribosomes washed in 10 -2 M.]~Ig2§ represents tRNA and about 2 . 8 ~ is due to 5 s rRNA (see Discussion). The molecular weight of B. emer~onii high molecular weight rRNA has not been determined. Therefore, to calculate the number of molecules of 5 s rRNA per ribosome we have made the following assumptions. First, we assume that these ribosomes are similar to those of other eucaryotes (Taylor & Storek, 1964). This appears to be the case, since the monomer form (75 s) dissociates into 63 s and 45 s particles when the ~Ig 2+ is lowered to 10-4 M. These values were obtained in sucrose gradients b y the method of ~[artin & Ames (1961) with SH-labelcd ribosomes of E. col/as the reference marker. The second assumption concerns the molecular weight of the rRNA of these ribosomes. There appears to be considerable disagreement as to the molecular weight of 28 s and 18 s rRNA in the literature. For the present calculations we have uscd the values of 0.63 • 106 and 1.6• 106 for 18 s and 28 s rRNA, respectively (Staehclin, Wettstein, Ours & Nell, 1964). Thus, we assume tlmt the total molecular weight of the RNA associated with 13. emersonii ribosomes is 2.3• 106 (5, 18 and 28 s). Since the molecular weight of 5 s rRNA is 51,000 (148 nuclcotides, see Results) and it represents 2 . 8 ~ of the total, then 21. emersonii ribosomes contain 1.3 molecules of 5 s rRNA per ribosome. Ribosomes isolated in 10 -2 ~t-]~Ig2+ contain about 1.7 molecules of tRNA Which are not removed by the washing procedure described. We reported previously (Comb, Sarkar, DeVallet & Pinzino, 1965) that B. emersonii ribosomes contained two molecules of 5 s rRNA (transfer-like RNA) per ribosome, but this was based on a lower molecular weight for 5 s rRNA than in the present TABLE 1

Distribution of transfer R N A and 5 s rRNA i~ the cytotglasm of Blastoeladiella emcrsonii

RNA

component

Transfer R I q A 5 s ribosomal R N A

Molecules per cytoplasmic ribosome Soluble ( 105,000 g Ribosome. supernatant bound solution) 5.1 nono

1.7 1.3

studies. Table 1 summarizes the data obtained on the distribution of tRNA and 5 s rRNA in the various cell fractions. I t should be noted that the cytoplasm of B. emersonii contains about 7 tRNA molecules per ribosome and about 25~/o of this is bound to ribosomes. E. cell cells contain about 10 tRNA molecules for every ribosome. (iii) Homogeneity of 5 s rRNA The question as to whether this RNA component represents a group of closely related molecules or a single species of RNA is of major concern in efforts to elucidate its biological function. Since no specific assay is available for estimating the purity of this component, we have attempted to answer this question by subjecting 5 s rRNA to the various chromatographic procedures that have been used to resolve partially closely.related species of tRNA. As demonstrated above (Figs l(b) and 2), a single sharp peak is obtained with 5 s rRNA from either organism on methylated albumin columns. We have never observed any indication of heterogeneity in this peak with

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Tubeno. Fzo. 4. The distribution obtained after a 200.tuba transfer of 5 s [a:P]rR~A in the automatic countercurrent distribution apparatus. In the solvent system used (see Materials and Methods) 5 s rRNA from B. emersonll displayed a partition coefficient (K) of 5.7. ( - - Q - - O - - ) 5 s [~:PJrRNA radioactivity; ( ~ O - - O - - ) theoretical. different salt gradients. On r e c h r o m a t o g a p h y of 5 s r R N A , a single peak is generally obtained. T On DEAE-cclluloso column c h r o m a t o g r a p h y at 80~ a single sharp peak is obtained. The best evidence we have t h a t 5 s r R N A is a single homogeneous species is its distribution after 200 transfers in the countercurrent distribution apparatus. Tiffs is shown in Fig. 4. The 5 s [s2P]rRNA used for this experiment was purified b y the procedure given above and represents the material from the pooled 5 s peak after a single methylated albumin column run. The observed distribution is v e r y close to the theoretical distribution for a single molecular species. The contaminating shoulder in the radioactivity profile represents less than 10% of the total. 0.8

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RNAconcentration(mg/ml.) Fro. 5. Comparison of the sedimentation coefficients of E. coil tR~A and 5 s rRI~'A as a rune9tion of 1ZNA concentration. ( - - O - - O - - ) 5 s rTClqA; ( - - O - - O - - ) tR~A. ,S'~ for tPA~'Aand 5 s rR~A 3.6 and 5"3, respectively. "[" Preparations of 5 s rRNA stored for extended periods at --20~0 sometimes yield a small second peak eluting at about the same salt concentration as the second peak in Fig. 8(b).

450

D . O . COMB AND T. Z E t I A V I - W I L L N E I ~ (b) Physical prol~erties of 5 ~ r R N A

(i) Sedimentation coegicient The sedimentation coefficients of t R N A and 5 s r R N A from E. coli as a function of concentration are compared in Fig. 5. This shows t h a t the scdimcntation of 5 s rRNA is much more dependent on concentration than tRNA, and suggests a more rigid, rod-like structure for 5 s rRNA than for tRNA. Extrapolation to zero concentration gives a sedimentation coefficient of 5.3 for 5 s r R N A and 3.6 for tRNA. (ii) Thermal denaturation of 5 S rRNA Figure 6 compares the heat denaturation curves for B. cmersonii t R N A and 5 s rRNA. Although the midpoint of the absorbancy rise (T~) is about the same for both species, the helix-random coil transitioI1 for 5 s r R N A is much sharper than that of

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60

80

FIn. 6. Thcrmal transition of B. emersonii 5 s rR~A and tl:th'A. Tim solution contained 0.15 .~[-~N'aC1,0.015 .~I-sodlumcitrate (pII 7) with and without; MgCl2 as indicated. The rncdian tcmpcraturo of tim helix-coil transition (Tin) ia the abscnco of Mgz§ for both I=tN'Acomponents was 57~ ( - - O - - O - - ) 5 s rR~A; (--@--@--) tRNA. tRNA. One possible interpretation of these data is that 5 s r R N A contains one or more helical regions with similar G-O or A - U base pah'ing. I n contrast, the broader tra1~sition of t R N A suggests helical regions of varying base composition which melt independently (Felsenfeld & Sandecn, 1962). I n the presence of magnesium ions, the helical regions of both species are stabilized. (iii) Chain length of 5 s r R N A The method of 3fidgley (1965), modified, was employed. The 2'- and 3'-hydroxyls at the 3' terminus were oxidized and one of the two moles of aldehyde formed were treated with 14C-labeled isonicotfific acid hydrazide to form the hydrazone. The labeled RNA was chromatographcd on methylated-albumhl columns so that a more accurate estimate of the chain length throughout the absorbancy peak could be obtained. The results of these studies are sho~m in Figs 7 and 8. Two controls were run. First, t R N A was treated with [x4C]isonicotinic hydrazide without prior pcriodate treatment. Figure 7(a) demonstrates tlmt no detectable radioactivity is associated

5 s RIBOSOMAL 0.2

I~NA

451

No ]0~ (control) (a) tRNA

800

0.1

::I.

400 .~_

O ~o

u (b)

tRNA

9

,~ 0-2

X

9

X

x X

Average

x /ht

chain

~

length

X

t,ooio

1200

S0 u

X

800

0-I

400

30

!

!

40 Tube no.

50

Fro. 7. Chaln-length dctermination of B. emersonii tRXA. Methylated albumin column chromatogram of t R N A treated with [x~O]isonleotinie aeld hydrazido (a) without periodato oxidation (control) and (b) after oxidation with periodate. Tho details of the reaction and chromatography aro given under Materials and Methods.

(--@--@--) Absorbaney; ( - - O - - O - - ) ets/min. Tho column sizo was 1 cm x 26 era.

with the t R N A peak. Tile second control was simply a cheek on the method to see if the proper chain length is obtained ~ i t h tRNA. I n Fig. 7(b) the chain length has bccn plotted from the absorbaney and radioactivity of each tube. A chain length of 77 • 3 nucleotides was obtained over the main absorbaney peak of tRNA. These results confirm Midgley's studies and indicate that the method sholfld be applicable to 5 s rRNA. I t should be pointed out t h a t the method is o,fly useflll in those cases where the 3' terminus is unesterified. R N A degraded b y RNase would contain a 3' phosphoryl group and would not react with perlodate. Figure 8 demonstrates the use of this technique ~ t h E. coli and B. emersonii 5 s rRNA. Two absorbaney peaks are obtained when either preparation is c h r o m a t o ~ a p h e d oR methylated albumin columns. Since freshly isolated 5 s rRNA yields a single peak upon rcchromat o ~ a p h y on methylatcd albumin columns (see also footnote on p. 449), we feel t h a t

452

D. G. COMB AI~D T. Z E I t A V I - W I L L N E R

0.21 (a)

x

X

x

210

X I X X XKX~ X A XX X X

190

!

Average choln length 195•

x

E. coil

0.1 =L E O ',O

--

1170

500

U

600

400 "2.

200

._= u

0

!

!

o

!

0

i

x x XxxXX 9 x

(b) m

~ x r~

< 0-4 B. emersonii

Average chain length

16oo

x

0"3

1200

0-2

800

0'1

400

40

u

J

I

50

60

70

Tube no.

Pzo. 8. Chain-length determination of (a) E. coli 5 s rR~A and (b) B. emersonii 5 s rlRNA. After oxidation of the terminal hydroxyls with pcriodato, followed by treatment ~ t h [l'O]isonicotinic acid hydrazido, the z40-labeled RN'A was chromatographed on a methylated albumin colurml. The specific activity of the [140]isonicotinic acid hydrazlde was 3100 cts/min/m~mole. ( - - t - - O - - ) Absorbancy; ( - - O - - O - - ) cts]min. The column size was 1 em • 26 cm. the two peaks obtained after modification of the end group represent a conformational change in the molecule. W h y E. coli 5 s r R N A is more susceptible to this conforma. tional change is not apparent at present. The results indicate a chain length of 148 nuclcotides for B. emersonll 5 s rRNA. The value of 195 nueleotides for E. cell 5 s rRNA, isolated from the peak shown in Fig. l(b) is too tfigh and suggests that about 25% of the 5 s r R N A molecules have an esterified hydroxyl group at the 3' ~erminus or that the preparation is contaminated with about 25% unrelated, unreacting polynucleotides. Prior treatment with alkaline phosphatase would resolve this problem, but this has not been done. Studies by 1qosset el al. (1964) and Schleieh & Goldstein (1966) have indicated a

5.s RIBOSOMAL

RI~A

453

Choln length 77 150 8 t

:L

I000

E 0

750 ~E

>- 0"3 o

n

..Q

u

< 0-2

/

0"1

40

soo

/

600 E

~-

400 -1-

50

60

200

70

Fraction no.

Fro. 9. Comparison of the eluted position ofE. coI~5 s rRI~A and tRNA and 5 s rR~A from B. emersoni~. E. coli 5 s rRN'A (10 A~s0 units) was combined with about 0.5 A=Go unit each of [3H]tR~A and 5 s [3~P]rR~A from B. emersoni~ and chromatographed on a DEAE-cclluloso column at 80~ (Materials and ]~Iethods).Since the chain lengths of the two isotope-labeled R~A components have been determined, we estimate that E. coli 5 s rRNA has a chain length of about 120 nucleotldcs. (~ X--• [aII]tI~A radioactivity; ( - - O - - O ~ ) 5 s [a2p]rRN'Aradioactivity; ( - - @ - - 0 ~ ) A260 E. cell 5 S rRNA. chain length of about 105 and 122 nucleotides, respectively, for E. cell 5 s rRNA. To demonstrate that the 5 s component from E. coli is smaller than the comparable species from B. enzersonii, E. colt 5 s rRN'A was ehromatographed with 5 s [a2P]rRNA and [3H]tRNA from B. emersonli on a DEAE-celluloso column at 80~ Figure 9 demonstrates that E. coil 5 s is eluted about 0-6 of the distance between molecules with 77 nucleotides and 150 nucleotidcs. We do not know ff the elution position of molecules this size is strictly dependent on chain length, but the results do suggest that E. cell 5 s rRNA is smaller than B. emerson~ 5 s rRNA, and we tend to agree with the results of others that the chain length is approximately 120 nueleotides. (e) Base composition of 5 ~ r R N A The major nucleotide composition of 5 s rRb~A from E. cell is Up, 22.4; Gp, 31-6; Cp, 28.4 and Ap, 17-6; and from B. emersonli, Up, 23.7; Gp, 32.5; Cp, 24.6 and Ap, 19.2. These values are very similar to the major nueleotide composition of tRb~A from the respective organisms. (i) Methylated base content of 5 S r R N A The one strildng difference between tRNA and 5 s rRNA is the absence of detectable methylatcd bases in the latter. Figures 3 and 10 compare the radioactivity due

D. G. COMB AND T. Z E H A V I - W I L L I ~ ' E R

454

(a)

(b)

•0.15

tRNA

O

5s rRNA

I

~ooo

o.Io

t

16s+ 23s

8

9

soo

~: o.o5

J 30

40

40

50

50

70

Fraction no.

Fro. 10. Mcthylated base content of E. coli 5 s rRN'A. The methylatcd albumin column chromates,crams orE. co~i (a) tR~A from the 105,000 g supernatant solution and (b) 5 s rRNA extracted from the ribosomes are shown. The cells were given a 10-min pulse of [140-methyl]methlonine prior to isolation of the R~A. The radioactivity profile shows the distribution of I'r containing methylatcd base. The eel,ran size was 1 cm • 26 cm. ( - - O - - O - - ) Radioactivity; ( - - O - - Q - - ) absorbancy. to mcthylated bases in t R N A and 5 s rRRTA isolated from B. ernersonii and E. col{ after in vlvo exposure to [140-methyl]methionine. I t is clear from these results that radioactivity due to methylated bases is not associated with the main absorbancy peak of 5 s r R N A from either organism. The radioactivity at the leading edge of the 5 s peak is due to t R N A contamination, l~rom the specific activity of the t R N A ig is possible to estimate t h a t contamination of 5 s r R N A is less than 5% in both eases. Studies not reported here have demonstrated t h a t the radioactivity associated with the I~NA is due to methylated bases, and not amino aeyl I~NA or incorporation of label from the one-carbon pool into the purine or pyrimidine rings. (ii) Pseudouridine content of 5 s rt?NA To estimate the pseudouridine content of this t%NA component, B. emersonii was grown on [2-14C]uraeil and the 5 s r R N A peak isolated b y the above procedure. To remove as much contaminating t R N A as possible, all but the leading edge of the 14C.labeled 5 s peak was pooled and rechroraatographed on a methylated albumin column. RI~A from the tubes, indicated b y the arrows in rig. 11, was isolated and analyzed for total radioactivity associated with uridine and pseudouridine as described under BIaterials and ~Iethods. The radioactivity profile associated with each base in the paper chromatograms is also shown in Fig. ! 1. R N A from the leading edge of the 5 s peak contained 0.70 mole of pseudouridine per molecule and from the trailing edge 0.57 mole per molecule. I n similar experiments it was determined t h a t t R N A contains an average of 2.4 moles per molecule, and lfigh molecular weight r R N A contains 1.5 moles of T per 100 nucleotides. I f the pscudouridinc in 5 s r R N A was due to contamination, it would require 10% contamination with t R N A or 30% contamination with rRNA. Since we have determined from [140]mcthylated base content t h a t the trailing edge of the rechromatographed 5 s peak contains less than 1% contamhmtion with tRNA, it seems likely t h a t the pseudouridine content of

,5 s I~IBOSOMAL B.l~'h

A

455

B

0

~0.I0

o.os

......

d~____J

.......

20

---

v 40

30

Fraction no.

9~-400

.~

3oo

,oo

U !

1

!

!

3

!

!

5

!

!

| I I I I I I

7 ! 3 Paper strip no.

5

7

:Fro. 11. Determination of tlm pseudouridino content of B. emersonll 5 s rRI~A. Top: Absorbancy profile of 14C-labeled 5 s rR~XA (68,000 cts[min/A26 o unit, labeled in viva with [HC]uracil} aftcr rcchromatography on a methylated albumin column. Tho arrows (A,B) indicato the singlo fractions used for tho estimation of pseudouridine contcnt of tlm RNA. Bottom: Separation of uridino and pscudouridino 04C-labeled) by descending paper chromatography in water-saturated butanol for 48 hr. The paper was cut into strips as indicated and counted in a scintillation spectrometer. Tho percentage of tho total counts (ku plus U) present in pseudouridino at each sido of tho RNA peaks aro indicated. A tracing of tho ultraviolet-absorbing spots on tho chromatogram is shown at tho bottom. 5 s rR1VA is real a n d t h a t a b o u t one-half the molecules c o n t a i n one pseudouridine residue. (fii) Thiopyrimidine content of 5 ~ r l l N A W e have e x a m i n e d tile a b s o r p t i o n s p e c t r u m of tiffs R N A species from B. emersonil i n the 300 to 340 m/z region, and find n o evidence for a peak a t 330 m/~ as dcscribcd b y Lipsett (1965) for I~NA c o n t a i n i n g 4-thiouridylic acid. (iv) 5' terminus of 5 s r R N A To detcrmine if the 5' t e r m i n u s contained a n csterificd phosphoryl group, the R N A from ]3. emersonii was i n c u b a t e d with pol)naucleotide kinase a n d y-labeled [32P]ATP before a n d after t r e a t m e n t with alkaline phosphatase (Richardson, 1965). Before alkaline phosphatase t r e a t m e n t , 3290 ets/min/A26 o u n i t wcrc incorporated into the

456

D. G. COI~IB AND T. Z E H A V I - W I L L N E R

molecule, whereas after removal of phosphoryl groups 26,850 cts/mln/A26o unit were incorporated. This indicates that 88% of the molecules contained a 5' phosphoryl group. This observation, along with the fact that the 3' terminus is not esterified, indicates that 5 s rRNA probably does not arise by RNase cleavage of high molecular weight RNA at either end of the molecule; since if 5 s arose by RN'ase cleavage near the 5' terminus, then a 3' phosphoryl group would be expected, and if cleavage occurred near the 3' terminus, there should be no 5' phosphoryl group in 5 s r R ~ A . 4. D i s c u s s i o n 9The 5 s rRNA isolated by the procedure described appears, b y column chromatography and countercurrent distribution, to be homogeneous. A single uniform peak is obtained by chromatography on either a methylated albumin column or a DEAEcellulose column a t 80~ Countercurrent distribution of 5 s rRNA in the solvent system used in the past for tRNA yields a single peak with a distribution close to that predicted for a single solute. The final preparation used for these studies contained less than 5% tRNA. The chain length of B. emersonii 5 s rRNA was determined to be 148 nucleotides and, therefore, it would have a molecular weight of 51,000. The method employed depends on an uncsterified 3' terminus, and we have no independent evidence that all the molecules are in this condition. This estimate is a maximum value; but from comparative studies with E. cell 5 s rRNA, it probably represents a very close approxlmarion. We have attempted to estimate as precisely as possible the total amount of 5 s rRI~A on the ribosome. This was done by measuring the area under the various RI~A peaks in methylated albumin column chromatograms. An average value of 2.8% of the total rRNA was obtained for 5 s rRNA in six different preparations of B. emersonii ribosomes. A value of 3.0% was obtained for two separate preparations of E. coli ribosomes. This amounts to slightly more than one molecule per ribosome and agrees with the previous estimate of I~osset et al. (1964). The results of in rive labeling with [14C.raethyl]methionine clearly demonstrate that 5 s rRNA from either organism does not contain methylated bases. This is a distinctive characteristic of this component, since tRNA and high molecular weight rRNA contains a variety of methylated bases. Our estimate of about 0.5 mole of pseudouridine per molecule of 5 s rRlgA is of interest but without explanation, since the function of this unusual base in other RNA molecules is unknown. Since conversion of uridine to pseudouridine may occur at the polynucleotide level (Weiss & Legault-Damare, 1965), the presence of less than one mole] per molecule may indicate a transitory state in the function of this molecule, or it m a y indicate that 5 s rRNA is composed of two distinct but very similar species. When 70 s ribosomes of E. colt are dissociated in 10 -4 )[-l~Ig2+, the 5 s component remains attached to the 50 s subunits (Rosset et al., 1964). A similar situation probably occurs in B. ernersonil, hut because of the difficulty in separating the large subunit from dlmerized small subunits in sucrose gradients, this relationship is not so easy to demonstrate. I t is clear, however, that 5 s rRI~A has a very high affinity for the ribosomes, and we have been unable to remove it by simply lowering the Mg2+ concentration or by dialysis of ribosomes in the absence of l~Ig2+. When B. emersonii ribosomes are treated with EDTA, the 5 s component is released without loss of protein but the ribosomes undergo some sort of conformational change to yield 50 s and 30 s

5 s I~IBOSOMAL I{I~'A

457

particles which we have been unable, so far, to convert to normal 75 s particles when Mg2 + and 5 s r R N A are restored. These observations, as well as studies on the binding of 5 s r R N A to ribosomes, will be discussed in a subsequent paper. The methods commonly used for the extraction of tRN'A from either whole cells or from extracts of cells containing ribosomes will also extract the 5 s rRN'A. Since 5 s rIClgA amounts to about 3% of the total cellular RNA, and tI~N'A about 10 to 15%, preparations of t R N A prepared ia this manner will contain a significant proportion of contaminating 5 s rRN'A. One final point: several investigators have recently suggested t h a t 5 s r R N A m a y represent degradation products of either messenger RN'A or rRNA. We feel t h a t this is lfighly unlikely for the follo~ing reasons. I t is much too homogeneous with respect to chain length, secondary structure and 3' and 5' termini to be a random degradation product. E . cell 5 s r R N A appears to have primarily uracil at the 5' and 3' ends of the molecule (Rosset etal., 1964; Schlcich & Goldstcin, 1966). The absence of methylatcd bases and the very low content of pscudouridine essentially exclude the possibility t h a t it arises by random degradation of mature high molecular weight rRN'A. :Furthermore, 16 s and 23 s rRN'A do not compete with 5 s r R N A for complementary sequences on DNA (Zchavi-Willner & Comb, 1966). T h a t 5 s r R N A represents a random degradation product of messenger R N A also seems unlikely for the following reason. During the blastula stage of sea urchin development, active incorporation of isotope into t R N A and messenger I~NA occurs. However, there is no detectable incorporation of isotope into the 5 s rI~NA even after a long chase of pulse-labeled RNA. The first detectable synthesis of 5 s r R N A in the embryos occurs much later in development, when high molecular weight rI~NA is first synthesized. The function of 5 s rl~R'A on the ribosome is far from clear. However, recent studies in this laboratory indicate t h a t one molecule of 5 s r R N A binds specifically (not displaced by tRNA) to the larger subunit at either 10 -2 or 10 -4 :~I-l~Ig2+ concentration. However, when both subunits are present and they associate to form the monomer, no specific binding or exchange of 5 s r R N A occurs. This suggests that the binding site m a y be located at the point of contact between subunits and that 5 s r R N A m a y be involved in their association. ~Vo actmowledge the expert technical assistance of l~Ir Jose DoVallet and are grateful to Dr Charles C. Richardson for carrying out, in part;, the polynuclcotido kSnase studies. This study was aided by grant no. GM 12632 from the National Institutes of :Health of the United States Public :I-Iealth Service and grant no. GB 23~9 from the National Science Foundation. One of us (D. G. C.) was a Research Career Development awardeo of the United States Public tIealth Service, 1-K3-GSI 6385. rCEFERENGES Apgar, J., :Holley, R. W. & l~Ierrill, S. :H. (1962). J . Biol. Chem. 237, 796. Bray, G. A. (1960). Analyt. Biochem. 1, 279. Brown, D. D. & Littna, E. (1966). J. Mol. Biol. 20, 95. Cantino, E. C. & :Horenstein, E. A. (1956). l~Iycologla, 48, 777. Cantino, E. C. & L o w t t , J. S. (1964). In Advances in Morphogenesis, cd. by 1~I.Abcrcrombie & J. Brache$, vol. 3, p. 33. Now York: Academic Press. Comb, D. G., Brown, It. & I~atz, S. (1964). J . l~lol. Biol. 8, 181. Comb, D. G. & Katz, S. (1964). J. Mol Biol. 8, 790. Comb, D. G., Katz, S., Brands, it. & Pinzino, C. J. (1965). J. Mol. Biol. 14, 195. Comb, D. G., Sarkar, N., DeVallet, J. & Pinzino, C. J. (1965) J. Mol. Biol. 12, 509. FcIsenfeld, G. & Sandeen, G. {1962}. J. Mol. Biol. 5, 587.

458

D . G . COMB AND T. Z E H A V I - W I L L N E R

Galibert, F., Larsen, C. J., Lelong, J. C. & Bolron, 1~I. (1965). Nature, 207, 1039. Goldthwait, D. A. & Kerr, D. S. (1962). Biochim. biophys. Acta, 61, 930. Hayward, 1%. S., Legault-Dcmare, J. & V~reiss, S. B. (1966). Fed. Prec. 25, 520. Katz, S. & Comb, D. G. (1963). J. Biol. Cfiem. 238, 3065. Lipsett, ~I. N. (1965). J. Biol. Chem. 240, 3975. l~Iandcll, J. D. & Hershey, A. D. (1960). Analyt. Biochem. 1, 06. l~Iarcot-Quciroz, J., Julicn, J., Rosset~ R. & ~lonler, 1~. (1965). Bull. Soc. Chim. Biol. 47, 183. l~Iarmur, J. & Dory, P. (1962). J. 11Iol. Biol. 5, 109. l~Iartin, R. G. & Ames, B. •. (1961). J . Biol. Chem. 236, 1372. ZIidgley, J. E. ~I. (1965). BiocMm. biophys. Acta, 108, 340. Richardson, C. C. (1965). Prec. Nat. Acad. Sci., Wash. 54, 158. Rosseb,/~., ~Ionier, R. & Julien, J. (1964). Bull. Soc. Ghlm. Biol. 46, 87. Sarkar, ~ . & Comb, D. G. (1966). J . lllol. Biol. 17, 541. Schleich, T. & Goldstcin, J., (1966). J..Mol. Biol. 15, 136. Staehelin, T., ~Vettstein, F. O., Oura, H. & ~oll, H. (1964). 1Vature, 201, 264. Taylor, ZI. ZI. & Storck, R. (1964). Prec. Nat. Acad. Scl. Wash. 52, 958. ~Veiss, S. B. & Legaulb-Dcmare, J. (1965). Science, 149, 429. Zehavi.~Villner & Comb, D. G. (1966). J . Mol. Biol. 16, 250.