Small Ribonucleic Acids of Escherichia coli

Escherichia coli RNA molecules in the size range of 70 to 400 nucleotides have been characterized by polyacrylamide gel electrophoresis and by RNase T, (EC 2.7.7.26) fingerprinting. RNA labeled by 32P043p for 20 min is separated into about 20 bands by electrophoresis in 5, 10, and 20% polyacrylamide gels; many of them represent pure RNA species. Some correspond to known molecules such as tRNAs, 5 S RNA, 4.5 S RNA, and 6 S RNA, but others have not been described previously. The amounts of these latter molecules are low, usually less than 10% of the level of 5 S RNA. One molecule, with electrophoretic mobility similar to 5 S RNA, has a 5’-terminal guanosine triphosphate. Another, with a moblity similar to that of 6 S RNA, contains dihydrouracil. Several different RNA preparation methods give essentially the same results. Two-dimensional polyacrylamide gel electrophoresis (10 % --f 20 % polyacrylamide) was used to purify RNA in the 4 S to 5 S region. This technique is capable of resolving at least 30 different RNA species ; many of them are pure tRNA molecules. It allows for direct comparison of the quantities of purified tRNAs and the other molecules described in this paper. Such analysis shows that during 20 min’labeling the newly characterized molecules accumulate at levels comparable to many individual tRNAs.

coli RNA molecules in the size range of 70 to 400 nucleotides have been characterized by polyacrylamide gel electrophoresis and by RNase T, (EC 2.7.7.26) fingerprinting.
RNA labeled by 32P043p for 20 min is separated into about 20 bands by electrophoresis in 5, 10, and 20% polyacrylamide gels; many of them represent pure RNA species.
Some correspond to known molecules such as tRNAs, 5 S RNA, 4.5 S RNA, and 6 S RNA, but others have not been described previously.
The amounts of these latter molecules are low, usually less than 10% of the level of 5 S RNA.
One molecule, with electrophoretic mobility similar to 5 S RNA, has a 5'-terminal guanosine triphosphate. Another, with a moblity similar to that of 6 S RNA, contains dihydrouracil.
Several different RNA preparation methods give essentially the same results. Two-dimensional polyacrylamide gel electrophoresis (10 % --f 20 % polyacrylamide) was used to purify RNA in the 4 S to 5 S region.
This technique is capable of resolving at least 30 different RNA species ; many of them are pure tRNA molecules.
It allows for direct comparison of the quantities of purified tRNAs and the other molecules described in this paper.
Such analysis shows that during 20 min'labeling the newly characterized molecules accumulate at levels comparable to many individual tRNAs. fLs~herichia coli ~11s coiitaiii a i~ufnbc~r of IISI1 molecules that havr lxwl rli:lixcterized by fullctioii or structure. I\folecules such as trnnsfrr RNAs and rihosonlal RN&Is have bee11 well stud& from both viewpoints (l-4). This has becu possible brrnuse of thrir relative abulidancc in the rcll :ultl t.heir defined J~l~ysiologicxl roles. Structural studies of some other RNA 1iat.r l:ggytl brhiiid functional studirs, oltelr brcausc of difficultirs ilr obtairlitlg sufficient arnoulrts of J~urc material.
011r ol these probably corresporids to a molecule that Goldstein and I-lni~ewood (15) isolated by Sephndes chromatography and polyacrylarnide gel electlophowsis.
.lluleriuls purchased from Eastman Organic Chemicals. Acrylamide (catalogue Number 5521) and N ,N'-methylene-bisacrylamide were also obtained from Eastman Organic Chemicals and used after filtration of a solution (usually 38% acrglamide, 2y0 bisacrylamide in glass-distilled water) through Millipore filters (HAWP04700).
Fluoroglide for fluorocarbon coating (S9020) was obtained from Scientific l'roducts, Evanston, Illinois, and urea (catalogue Number 9200) was from Schwarx-Mann 13iochemicals, Orangeburg, N. Y. All test tubes and centrifuaation tubes were siliconized (rinsed in 1 % solution in toluene of dichlorodimethylsilane from Aldrich Chemicals, Milwaukee) and baked 3 hours at 150". All heatstable reagents which were used during sample 1xel)aration were autoclaved at 115" for 20 min. Carrier RNA, purchased from Sigma TGochemicals, was llhenol extracted at 80" and ethanol prccipitatcd before use.
Under these conditions the generation time is 1 hour.
When the cell density reached 1.5 to 2.5 X lo* bacteria I)er ml, the cultures were labeled by the addition of neutralized 3211043P to a final concentration of 0.3 mCi l)er ml. After 20 min labeling, 3~l'~labeled RNA samples were l)rel)ared by one of the following four different m&hods.
The mixture was immediately shaken for 15 min at room temperature.
The supernatallt was saved, and the phenol phase was extracted with 0.5 ml of 100 nm Tris-HCI (pH 7.5) for 5 min. After the second celltrifugatioll, both supernatants were combined and poured into 6 ml of 95% ethanol containing 0.25 ml of 2 M sodium acetate (PH 5). After storage at -20" overnight, the lxecipitated RNA was collected by celltrifugation at 10,000 X g (O-2") for 20 min and resuslxncled in 2 ml of 300 mM sodium acetate (pH 5). These ethanol precipitation procedures were repeated three more times before electrophorcsis.
.l/ethod 2-This method is based on the hot SDS1 treatment described by Uremer and Yuan (23). After V labeling, the culture (2 to 20 ml) was poured into an equal volume of hot sodiunl dodecyl sulfate solution (1 To SDS, 200 rnrvf sodium chloride, 40 mtil EDTA, and 20 mM Tris-IICl, l>H 7.5) which had been kept in a boiling IT-ater bath. Ninety seconds later, the clear lgsate was cooled to room temperature.
Diethyll)yrocarbonate was added to :I final concentration of O.l%, as well as carrier RNA (to nlake 25 wg per ml) and potassium phosphate buffer pH 7.0 (to make I .5 mu).
The samljles were shaken at 37" for 10 min.
After addition of sufficient concentrated sodium chloride to make the samples 0.4 M sodium chloride, the samples were chilled in an ice bath for 10 min and then cclltrifugcd at 10,000 X g (O-2') for 15 min. The supernatant fluid was poured into 2.5 times its volume of ethanol and stored overnight at -20". After celltrifugation (10,000 x g, 0.2", 20 min) the RNA precipitates were resuspended in 2 ml of 0.25% SIX, 50 rnh1 sodium chloride, 10 mM EDTA, and 5 mM Tris-HCl, pH 7.5, and extracted at room temperature with an equal volume of buffer-saturated phenol. The RNA was then precipitated twice with ethanol from (0.5 to 1 .O ml) 300 m&f sodium acetate (pH 5).
Method S--In this method, the culture was poured on an equal wright of crushed ice containing botlium azide (1.5 rng per g of ice) and centrifuged at 15,000 x g (O-2") for 3 min. The cell pellet was suspended at O-5" in medium containing 1 mM sodium azidc and then poured into the hot SDS solution as in Method 2.
Jlethod &This was a modification of Xlcthod 2 including mild alkaline treatment to dcacylatc peptidyl-tRNA. The 2-ml culture was poured in a hot SDS solution of different composition (100 mM sodium chloride, 1% SIB, and 8 mM IIl)TA, pH 7.0) according to l\lethod 2. After cooling, 1 ml of 1 M sodium carbonatc buffer was added to obtain l)H 10 in the final solution, which was then incubated at 37" for 30 min (24). Ten milliliters of ethanol were added, colltaillina sufficient HCI to make a neutral fillal solution (about 0.1 ml of concentrated HCI).
The RNA was precipitated by ethanol and purified by 1)henol estraction as in Method 2.

Polyacrylamide Gel Electrophoresis
Electrophoresis on polyacrylamide gel blab (17 cm long X 13 cm wide) was done in the vertical cell E-C470 (E-C Apparatus, St. Petersburg, Fla.), according to the procedure of Peacock and Dingman (25)) with a few modificatiolls. TTnless otherwise noted, the electrophoresis buffer was Buffer A (25) which COW i;ists of 21.6 $ of Tris base, I .86 g of disodium EDTA, and 11.0 g of boric acid per liter, giving a final pH of 8.3. Buffer w>is recirculated between the top and bottom buffer reservoirs during clectrophorcsis.
In the case of 5% or 20% gels, the concentrations of acrylamide and N ,N'-methylenebisacrylamide were halved or doubled, resprctively. IMore application to the gel, the RNA samples (about 100 pg-) were heated to about 60" for 3 min in 10 ~1 of a solution containing 50% urea, 25 mM sodium chloride, and 4 IIIM EDT;1 After cooling to room temperature, the Farnplcs were mixed \vith 10 ~1 of a solution containing 20(7; sucrose, 1 rnM EDTA (pFI 7), and 0.1% bromphenol blue, and applied to the gel. During electrophoresis (at about 10 volts per cm) the cells were cooled by circulation of coolant maintained at 15". Esscntiall\. the same results were obtained with or without heat and urea treatment. When eluted samples were further purified by re-elcctrol)lloresis they were heated to about 80" in urea, as above, before application to the second gel.
When 20% gel was used, a few modifications were introduc*etl. Because the 20y0 gel sticks very tightly to the l'lesi&~s of the electrophoresis apparatus, it was necessary to coat \\-itli fluorocarboll both the slot former and a border of about 2 cm around the two sides and bottom of the (Boolant plates that are in direct contact with the gel. Unless the cell is treated in this way, it is very difficult to remove the slot former after polymerization or to fluorocarbon coating, however, it was occasionally necessary to apply rather strong force during dismantling.
The polymerization of the 20% gel was done while coolant was circulating at 15", to prevent the trapping of gas bubbles between the gel and cell surfaces. This type of bubble distorts electrophoresis pa,tt,erils.
III two-dimensional polyacrylamide gel electrophoresis, the first dimension was carried out in a small slot (1.5 mm thick and 3.5 mm wide) of 1O7o slab gel at about 20 volts per cm for 3 hours in one-half diluted Buffer A. Samples were pretreated by heat and urea, as described above. After the run, a gel slice containing the 4 S and 5 S RNA region was cut out with a microtome blade.
The lateral position of the sample was determined frotn the positions of dye markers that were added to the sample slot at periodic intervals during the run. The gel slice was put into the electrophoresis cell at right angles to the first dimension in a procedure similar to that described by others for separations of some other RNA species (26)(27)(28)(29).
Twenty per cent acrylamide solution was poured into the cell and it was polymerized around and below the 10% strip.
Electrophoresis in the second ditnension was at about 20 volts per cm for 17 hours in one-half diluted Buffer A.

Azbtoradiography and Elution of RNA jrom Gel
After polyacrylatnide gel electrophoresis the RNA was located by autoradiography.
This was accomplished by placing the gel on a sheet of tnoist filter paper which was secured to a large piece of Saran Wrap in a thick paper folder.
A second piece of Saran Wrap was placed over the gel and the edges were sealed with tape. Spots of radioactive ink (321'043-added to fountaiI1-pen ink) were placed on the paper around the gel, for later alignment.
Often, before removal of the gel from the electrophoresis apparatus, the top 1 cm of gel was cut off and rernoved.
The lower part of the gel was then removed and briefly rinsed in water befort being placed on the filter paper. The top of the gel was theu placed in the corresponding position on the paper. This process greatly reduced the level of contamination of the gel caused by leaking of ribosomal RNA (16 S and 23 S), which cannot enter into the gel, out of the slot at the top.
The part of the gel corresponding to cnch RNA4 baud or spot was cut out and the radioactivity was measured by Cerenkov radiation of 32P (30,31). The piece of gel contaiuing the RNA was then ground with a glass rod in a centrifuge tube. After addition of 1 ml of 300 tnn1 sodium chloride containing 0.2c/, SDS, the gel suspension was shaken on a Vortex miser for 1 hour at room temperature.
The gel was pelleted by centrifugation at 4000 x g for 30 min and re-cst.ractcd two tnore times with 0.5 ml of the same solution.
The supernatants were pooled, 80 pg of carrier RNA were added, and RNA was precipitated by the addition of 5 ml of ethanol and overnight storage at -20". RNA was resuspended in 300 mM sodium acetate (pH 5) and reprecipitated two more titnes by ethanol before fingerprinting.
Fingerprint Analysis of RNA Two-dimensional fingerprints after RNase 'I'1 digestion of the RNA (1:20, w/w, enzyme to RNA) were made according to the tnethods developed by Sanger and his collaborators (12,13). Purified oligonucleotidcs in the fingerprint were analyzed by pancreatic RNase digestion after elution from the paper (10 ~1 of 0.1 mg per ml of enzyme and 2 mg per ml of carrier RNA in 10 mM Tris-HCI, pH 7.6; 37" for 30 min) and electrophoresis on l)EAE-cellulose paper as described by Adams et al. (32). Anal-ysis of minor bases iu tRNA was done by paper electrophorcsis and chrotnatography as described by Barrel1 (33).

Ten Per Cent Pobgacrglamide
Gel Electrophoresis oj" E. coli Small RNAs-I'olyacrylamide gel electrophorexis is an efficient technique for the separation of RNA molecules of similar sizes. We used this technique both to characterize and to purify the various small RNAs of E. co/i. Fig. In is an autoradiogram of a 10% polyacrylamide slab gel analyzing small E. coli RNAs labeled by 321'04a-for 20 min at 37". In order to test whether the gel pattern depends upon the tnethod of RNA preparation, samples were made by several methods in which cellular RNA metabolism was believed to be stopped rapidly.
The four methods which were used to make the following samples were described under "Experimental I'rocedure." Sample 1 was prepared by direct phenol extraction of the culture medium as described by Altrnan (22). Sample 2 was made by the hot SDS treatment described by Bremer and Yuan (23), in which the culture was poured directly into a hot SDS solution.
In Sample 3, the culture was poured on crushed ice containing sodium azide, and then centrifuged. The pclleted cells were then treated with the hot SDS solution. Sample 4 was prepared by a modification of that used for Sample 2, with the addition of mild alkaline treatment to remove aminoacyl and peptidyl residues from tRNAs (24). These four samples gave similar elcctrophoretic patterns. This is a good evidence that the RKA patterns arc not dependent on the specific method of RNA preparation.
The RNA in each numbered band was identified by RNase T1 fingerprint analysis. Band I RNA gave the very complicated but characteristic fingerprint of 13. coli tRNA mixtures which was obtained by Sangcr et al. (12). Hand I actually is resolved into at least two regions, containing different, tRNA species. This double band nature is cvideut only after very short esposurc of the autoradiogratn.
Band II also can be resolved into two parts, but again, this is best seen after short exposure.
The slow part of 13and II, called II', consists of two RN,4 species, as shown by further re-electrophoresis in a 13.5% gel (Fig. 2~). Roth of these RNA bands have minor bases such as dihydrouracil, thyminc, and pseudouracil.
The major, aud faster, part (called as II" in Fig. 2a) has the same fingerprint as tRNAF"" published by Dube et al. (19,34). The fingerprint of the minor, and slower, part of II' (IISS). is shown in Fig. 3~. This does not correspond to any published fingerprint.
Howcvor, compositional analysis of each of Tl oligonucleotide shows that this is tRNAfer which has recently been sequenced by Ishikura and Nishitnura.' The faster part of Hand II (called II" in Fig. la) contains an undertnet~hylated form of tRNAFeu and two or three other t&N&, as yet unidentified. We shall discuss the tnature and immature forms of tRNAteU m more detail in the accompanying paper (19).
The fingerprints of the RNAs in Bands III and IV were made after rc-elcctrophoresis in 11.5% or 13.5% gels, although the RNAs are essentially pure after the 10% gel electrophoresis. The fingerprint of Isand III RNA, shown in Fig. 3b is the same as that published by Griffin (17) for the 4.5 S RNA.
Therefore, we conclude that Band III RNA is 4.5 S RNA.
Griffin showed that this RNA does not have any minor bases. She also pointed out that. this tnolecule probably corresponds to one of the RN,4s which Hindley (14) described as being slightly stnaller thau 5 S RNA.
Fro. 1. Autoradiogram of polyacrylamide slab gel electrophoresis of "21'-labeled Escherichia coli RNA. a, a single slab of 107c polyacrylamide gel analyzing four different RNA samples prepared by different methods as described llnder "Methods." Time of electrophoresis was 8 hours. b and c, electrophoresis patterns The RNA of Band IV has a fingerprint which does not correspond to any published one (Fig. 3~). This RNA has no minor bases. Pulse-chase experiments described in the accompanying paper (19) showed that this RNA is unstable.
Sequence analy-sis3 of this molecule indicates that it has a triphosphate pppG-at the 5' end since one RNase T1 product co-electrophoreses on DEAE-cellulose paper at pH 3.5 with pppGp after alkali treatment, but with pGp after snake venom phosphodiesterase digestion.
The sequence at the 3'-OH end is U-U-U-U-U-U-A since pancreatic RNase digestion of the terminal oligonucleotide yields only Up, whereas snake venom phosphodiesterase produces p-4 and pU in the ratio 1:5; up to half the molecules lack the 3'.OH terminal i1. 3 RNAs of Bands V, V', and V" have the fingerprint of 5 S RNA, as reported by Brownlee and Sanger (13) Hindley (14) also observed a small separation of conformational isomers of 5 S RNA during polyacrylamide gel electrophoresis. In our experiments, the relative ratios of the three isomers, especially the ratio of V" to the others, is variable.
We would like to stress the fact that we find no evidence of similar conformational isomers for any other small RNAs.
The fingerprint of the RNA of Band VI is shown in Fig. 3d. This is the fingerprint of 6 S RNA obtained by Brownlee, who determined the primary sequence of this molecule (16). Analysis of each T1 oligonucleotide by pancreatic RNase digestion confirms this conclusion.
Brownlee (16) noted that this 6 S RNA corresponds to the one described both by Hindley (14) and by Goldstein and Harewood (15). The RNA in a weak band, VI', has a fingerprint only slightly different from that of the 6 S RNA in Band VI. The differences, as well as radioactivity pulsechase experiments (19), indicate that the RNA in Band VI' is most likely a precursor of 6 S RNA.
These two correlated molecules are not heat interconvertible.
In the region between 5 S and 6 S RNA, there arc at least three distinct RNA molecules, although they can barely be seen in Fig. la because of their low intensities.
The wide region between Bands V' and VI was eluted and repurified in a 11.5% gel after gel. a, re-electrophoresis, in 13.57; acrvlamide, of Band IIs from the lO70 gel shown in Fig. la. b, repurification, in 11.5y0 polyacrylamide; of RNA obtained from the ree?on between V' and VI (Ficr. la). c. the rerrion containing mature tRNA?" in a 207, polyacr~lamide gel (Figrlc) which ha; been determined both by co-electrophoresis with mature tRNAp" and by fingerprinting, was repurified in a 10YP polyacrylamide gel with a marker of pure undermethylated tR,NAp. The undermothylated tRNAp migrates slightly faster than the mature tRNAp in 10% gel, as mentioned in the text.
heat-urea treatment. Fig. 2b is an autoradiogram of such a re-electrophoresis.
Five different bands can be observed. The RNA of Bands a! and 0 both give the 5 S RNA fingerprint and they presumably reflect a small amount of 5 S RNA contamination in the sample. RNA in Band y has a complicated fingerprint indicating inhomogeneity.
Although the RNAs of Bands 6 and E have simple and characteristic fingerprints (Fig. 3, e and f, respectively) they also are probably not completely pure at this stage; the autoradiographs of these fingerprints are of low intensity because of the low levels of these RNAs as seen in the gel. Band E RNA, which migrates just ahead of the 6 S RNA in 10% gel, contains the minor base dihydrouracil.
The accumulation of RNA 6 but not RNA E during stringent control conditions will be discussed in the accompanying paper (19).
In region VII of Fig. la there are a few very weak bands. Reelectrophoresis of the RNA in this region in 9% gel reveals at least three quite pure bands. The fingerprints of two of these bands, which have the fastest and slowest mobilities in the VII region, are shown in Fig. 3g (VIIF) and Fig. 3h (VII'), respectively.
Their intensities are very low, being only a few per cent that of 5 S RNA.
Five and 90% Polyacrylamide Gel Electrophoresis-In the region near the origin of the 10% gel patterns of Fig. la there are a few additional RNA bands. These bands are clearly resolved in lower percentage gels. The electrophoresis pattern of an E. coli RNA sample in 5yo polyacrylamide gel is shown in Fig. lb. The RNAs of both Bands VIII and X have rather complex fingerprints, indicating that they both are mixtures of two or more RNA species. Re-electrophoresis of each band in 7% polyacrylamide gel gives broad and partially separated bands. In contrast to the two Bands VIII and X, Band IX RNA has a relatively simple fingerprint (Fig. 3i). We have been unable to detect any minor nucleotides in this RNA.
Radioactivity pulsechase experiments show that this RNA is stable. From its electrophoretic mobility and the complexity of the fingerprint, we estimate that it is 300 to 400 nucleotides long.
Approximately 15 different small RNA species of E. coli are purified to a nearly homogenous stage by electrophoresis in 10% and 5yo polyacrylamide gels after re-electrophoresis in different concentrations of gel. However, there is only poor resolution in and near the bulk tRNA region.
In order to obtain better resolution of the RNAs in that region, 20% polyacrylamide gel electrophoresis was carried out. A typical electrophoresis pattern is shown in Fig. lc. The assignment of bands was made by RNase Tr fingerprinting.
The RNA band numbers corresponding to those in the 10% gel are shown in the figure.
The bulk tRNA region is resolved better here than on the 10% gel, but still only a few of tRNA species were found to be pure at this stage.
Several RNA molecules have rather unexpected mobilities in 20% polyacrylamide gels. 6 S RNA, which is 184 nucleotides long (16), co-migrates with the conformat,ional isomers of 5 S RNA which are 120 rmcleotides long (37). A second unusual feature is that the relative mobilities of the mature and immature forms of tRNAF"" are reversed from those seen on the 10% gel; as discussed above and elsewhere (19), in 10% polyacrylamide gel electrophoresis the immature form migrates faster, but in 20% gels the mature form migrates faster. In addition, the mature form of tRNAfe", which co-purifies on 10% gels with tRNAt"', migrates on the 20% gel free of tRNAi"' but with several different tRNAs. This is shown in Fig. 2c which is the 10% gel pattern of RNA that co-migrated with a marker sample of mature tRNAte" in the 20% gel. The mobility marker in the 10% re-electrophoresis is immature tRNAt"". RNA in Band w has the mature tRNATeU fingerprint, and the RNA of Band 9 has a fingerprint different from either (mature or immature) tRNAy"" or tRNAf"'.
Compositional analysis of the Tr oligonucleotides of this RNA (Fig. 3j) indicates that it is pure tRNAt"" which was sequenced by Blank and Sol1 (38).
of RNA in 4 S to 5 S Region- The preceding results show that changes of acrylamide concentration affect the relative electrophoretic mobilities of RNAs with similar sizes. Thus we concluded that two-dimensional electrophoresis should give good separation of RNA molecules, if the first and second dimensions were carried out in different percentage gels. Because of the strikingly anomolous behavior of RNA in 20 y. gels, relative to that seen in 10 y0 gels, we elected to use a 10% + 20% system, as described under "Experimental Procedure." Much of this procedure was adopted from the work of others in the purification of other RNA species (26)(27)(28)(29).
An autoradiogram of a two-dimensional polyacrylamide gel analysis of the small E. coli RNAs is shown in Fig. 4a. In this experiment, the region of 10% gel containing bulk tRNA and 5 S RNA was transferred to 20% gel for the second dimension. In the autoradiogram it is possible to see about 30 well resolved spots. Fingerprint analysis of each of the spots in this characteristic and reproducible pattern is now in progress.
Most of the spots represent pure RNA molecules, although several contain mixtures of molecules.
A schematic diagram of the two-dimensional gel is presented in Fig. 4b. The spots that contain four pure tRNAs, 4.5 S RNA, 5 S RNA, and Band IV RNA, are indicated by symbols placed above the respective spots in the dia-  In order to demonstrate the purity of the RNA in the two-dimensional gel spots, the fingerprint of one spot, marked Glu2 in this diagram, is presented in Fig. 3k. Analysis of this fingerprint indicates that this RNA is tRNATIU, which was sequenced by Ohashi et al. (39). After longer exposure of the gel (Fig. 4c) it is possible to see several additional spots. These spots, containing molecules that are slightly longer than normal tRNA, are indicated by the solid circles in Fig. 4b. Although their level is less than 1% of the amount of 5 S RNA, they give simple and characteristic fingerprints, indicating their homogeneity. Many of the RNA species were purified by electrophoresis in three different percentages of polyacrylamide gel, by successive re-electrophoresis, or by two-dimensional polyacrylamide gel electrophoresis.
The purified molecules were further characterized by RNase T1 digestion and fingerprint analysis.
Such analysis was also used as a measure of purity of a particular molecule, since inhomogeneous preparations have complex and irregular finger- prints in which the relative intensities of the different oligonucleotides are distributed over a wide range. However, purity as assayed by fingerprinting does not necessarily mean the existence of only a single species. Such conclusions are obtained only after determination of total primary sequence. For example, in a purified sample two molecules may exist which differ by only one nucleotide substitution or by minor differences at the 5' or 3' ends. Even such minor changes, however, often lead to noticeable mobility differences, giving either separate or oblong spots in the two-dimensional gel electrophoresis. Purification of individual molecules by re-electrophoresis of RNA mixtures is successful because mobility is determined not only by molecular weight but also other factors such as conformation. For example, conformation isomers of 16 S rRNA and 23 S rRNA have different mobilities and are clearly separated in appropriate concentrations of polyacrylamide gel (40,41). Thus, because of the complex nature of the separation, changes in electrophoresis conditions such as acrylamide concentration or temperature could have nonuniform effects on the mobilities of different RNA molecules with similar molecular weights.
Pre-liminary experiments of electrophoresis in 14 different concentrations of polyacrylamide gels (between 5 and 20%) confirmed these expectations.
As shown above, some RNA molecules which migrate together in one concentration of gel were well separated in another concentration.
There are two pieces of evidence which indicate t.hat the KPl'd pattern that we observe reflects the kinds of molecules in the cell. First, the bands probably do not result from RNA breakdown during isolation, since we obtain the same pattern regardless of the method of RNA preparation or the way in which ccl1 metabolism was stopped (Fig. la).
Of course, we cannot rule out a very fast acting nuclease that works in all cases before metabolism is completely halted.
However, we should point out. that 16 S rRNA, precursor 16 S rRNA (40,42), and 23 S rRNh can be reproducibly recovered in our RNA preparations with no evidence of degradation." Secondly, the bands are not degradat,ion artifacts created during electrophoresis nor are they composed of RNA aggregates, since the eluted RNA does not change its 1: FIN. 4. Separation of 32P-labeled RNAs by two-dimensional electrophoresis in polvacrylamide gel. The first dimension of electrophoresis was-in~lOG~~ polyacrylamide.
The region of that eel containing 4 S and 5 S RNA was subiected to further electrophoresis in 2o'r, polyacrylamide, at right "angles to the first dimension, as described under "Methods." The romarb rmumernls, arranged in the direction of first dimension, show the regions corresponding to the bands in the lo%, gel of Fig. la. a, an automobility during re-electrophorcsis in the same gel concentration even after heating in the urea solution.
The one exception is the set of 5 S RNA conformational isomers. The application of two-dimensional polyacrylamide gel electrophoresis to the study of E. coli small RNAs leads to the rapid purificat.ion of multiple RNA species. The results that we obtained using this more refined system agree very well with results we presented for one-dimension gels. Using this two-dimensional system it is possible to study molecules that are.similar in size to 5 S RNA or tRNA but which are much less abundant.
The use of the two-dimensional gel system to purify individual tRNA molecules also allows us to quantitate the absolute numbers of molecules, not just their biochemical activity. We can thus study both active and inactive tRNAs and compare them with other RNAs.
For example, it can be seen in Fig. 4 that several of the molecules under study hcrc are as highly labeled as many pure tRNA molecules.
Thus, they arc no more "minor RNA species" than are many tRNAs.
Finally, we wish to emphasize that there is no evidence to indicate that Band I contains only tRNA.
We expect that the two-dimellsiorlal gel elect,rophoresis system will be valuable in obtainiilg more information on this point.
Although this study is useful in describing a number of small RNA molecules other than tRNA and 5 S RNA, it has no direct bearing on the function of these other molecules.
Most of the radiogram of the gel. b, a schematic diagram of a. The positions of 5 S RNA, 4.5 S RNA, Band IV RNA, t,ItNA~'", tRNAp, tRNA?, and tHNA~"' are indicated in b. c, an autoradiogram of, the same gel, exposed for a longer time to permit visualization of the less intensely labeled spots. Electrophoresis was done in one-half diluted Buffer A and at the voltage of about 20 volts per cm in a gel. In this electrophoresis condition, Band IV RNA migrates with Band V in the 10% gel of the first dimension. molecules that we have studied were isolated on the basis of physical characteristics, such as discrete size or conformation. Since the method of RNA preparation does not seem to affect the pattern of bands on the gels, we feel that these bands are representative of the kinds of molecules that exist in cells as well as their relative rates of accumulation.
The actual numbers of various molecules per cell are not reflected in the intensity of the gel bands since we are studying relatively short labeling periods. This short term labeling does permit visualization of several molecules that are metabolically unstable (19) and have not been described previously.
Information about the location of these molecules within cells would undoubtedly be of great use in determining their functions. Characterization of these RNA molecules by polyacrylamide gel electrophoresis and fingerprinting should facilitate correlation of physical, chemical, and physiological studies. One such study on the accumulation of these RNAs wheu cells are in various physiological states, is presented in the accompanying paper (19).