Studies and Sequences of Escherichia coli 4.5 S RNA

a biologically stable species with electrophoretic properties between S and primary and behavior an stable

Its primary structure and behavior suggests an unusually stable and possibly unique secondary structure.
Even from single species of E. coli, there is some sequence heterogeneity within the molecule. The sequence of a major species from MRE 600 is:

Methods
for getting sequence overlaps on this highly structured RNA are described, and a possible functional role for 4.5 S RNA is discussed.
Escherichiu coli contains a number of low molecular weight RNA molecules for w-hich no function has yet been found. The best characterized of these is 5 S RNA which is known to be a component of ribosomes and was among the first RNAs to be sequenced (1). The sequence of another molecule, 6 S RNA, has been determined (2), but no definite function has been ascribed to it (3). A third such molecule has been designated as 4.5 S RNA (4) and studies on it, including the determination of its sequence, are described in this paper.
Studies on E. coli suggest that low molecular weight RNAs may be required for the initiation of an entire round of DNA replication (5) or as primers in its discontinuous mode of replication (6). Low molecular weight RNAs also have been implicated in the compact folding of E. coli DNA within a cell (7).
At this time, therefore, the paradox of fully sequenced molecules with no functions and functions with no fully characterized molecules exists.
Two recent studies show the existence of numerous other small RNA molecules in E. coli, but the evidence presented suggest they are more likely to be precursors of, rather than mature species of, RNA (3,8). Precursors of tRNA (9) and 5 S RNA (10) have been previously described and a precursor of * Present address, Imperial Cancer Research Fund Laboratories, P.O. Box 123, Lincoln's Inn Fields, London WCZA 3PX.
6 S RNA has been discovered (8). To date, no precursor of 4.5 RNA has been found.' 4.5 S RNA has been shown to be a component of a number of strains of E. coli studied by this author. It is found in the supernatant fraction, after sedimentation of ribosomes, and use of a, temperature sensitive mutant shows it to be a stable species. It is composed of 107 nucleotides and has no detectable minor bases. Its primary structure and properties indicate a G,C2 content similar to that of the transfer RNAs, a very high proportion of paired bases, and a stability greater than 20 kcal/mol. The results (in counts per min X 1O-3) for each species obtained from a single experiment are given in parentheses: t = 0, 6 S (3), 5 S (60), 4.5 S (12), tRNALeU (12); t = 60 min, 6 S (a), 5 S (23), 4.5 S (6), tRNALeU (6.5); t = 90 min, 6 S (4), 5 S (46), 4.5 S (12.5), tRNALeU   (8), showed that 4.5 S RNA was a stable species, and therefore an unlikely candidate for a simple tRNA precursor. Similar results have been found by Ikemura and Dahlberg (3). Preliminary attempts to charge 4.5 S RNA with an amino acid mixture were not successful. Careful examination revealed no minor bases in the molecule.

Sequence Analysis
Products of Complete Digestion with Ribonucleases-As a first step in sequence determination, the fragments obtained from complete digestion with either Tt or pancreatic A ribonuclease were separated, isolated, and quantitated and their base compositions and ultimately their sequences were determined. For the T1 fragments, the use of polyethylenimine-cellulose thin layer chromatography (Fig. 3) for a second dimensional separation was particularly valuable. This technique separated the three T1-octanucleotides found to be present in 4.5 S RNA (ionophoresis on DEAE-cellulose paper (Fig. 7) did not) and resolved the 3' end oligonucleotide (which streaked badly on DEAE-cellulose paper) as a discrete spot. The sequences of the small oligomers obtained by complete digestion with ribonucleases are summarized in Table II. In all cases, the 5'-terminal nucleotide in a sequence was determined by comparing the composition of a complete venom phosphodiesterase digest with that obtained either from alkali or Ts-ribonuclease.
In many cases, this, together with T1 plus pancreatic ribonuclease A digestion, gave sufficient information to order the sequence. The legend to Table II shows the other methods and enzymes used.
Additional elaboration was necessary in a few cases, however, to complete the sequences.

FIG. 1 (top Left
). An autoradiogram of the separation of low and in the second by thin layer chromatography on a sheet (20 X molecular weight s2P-labeled RNA from Escherichia coli by gel 40 cm) of PEI-cellulose (4). For the second dimensional separaelectrophoresis. A slab gel (20 X 40 cm) composed of 10% acryl-tion, the thin layer chromatography plate was eluted up to 10 cm amide/0.5%bisacrvlamide was used and electrophoresis was carried out at 4" using borate buffer, pH 8.3 (12). The position of using 1.5 M pyridinium formate, then transferred to 2.2 M pyridinium formate and eluted up to 30 cm. This step-wise technique 4.5 S RNA relative to other low molecular weight RNAs and to a bromphenol blue (B) dye marker was determiied by autoradiog-allowed for maximum separation of the larger oligonucleotides without loss of resolution of the smaller oligonucleotides. The raphy; one-half of the gel is shown. Gel bands were excised and identification of each nroduct is shown. 4.5 S RNA from Eschecounted by Cerenkov radiation. From a number of similar experi-richia coli CA 265 gave, in addition to products shown, another ments, the yield of 4.5 S was found to be roughly equivalent to that molar equivalent of C-A-G. B is the position of the xylene cyan01 of a single species of transfer RNA. blue dye marker. FIG. i (top right). An autoradiogram of the separation by gel electronhoresis of low molecular weight 32P-labeled RNA isolated from J&cherichia coli cells during early (E) and late (L) log phase growth. The gel conditions used-were-the same as those described in Fig. 1. At E the Ars, of the cells was 0.40; at L Asjo was 1.0. The yield of 4.5 S RNA relative to tRNALeU '(17) was ielatively constant with time, whereas the yield of 6 S RNA increased, being much greater at late than early log phase.
FIG . 3 (bottom left). An autoradiogram of a two-dimensional separation of the &onuclease T1-terminal digestion products of 32P-labeled 4.5 S RNA from MRE 600. Senaration in the first dimension was by electrophoresis on cellulose acetate, pH 3.5 (14), In the cases of the T1-octanucleotides U-U-U-A-C-C-A-G and U-C-A-C-U-C-U-G, it proved necessary to cleave the molecules with Uz-ribonuclease and separate the products (U-U-U-A, C-C-A, G, and U-C-A, C-U-C-U-G, respectively) before definitive sequences could be determined.
In the case of U-U-C-U-C-C-C-G, the CMCT modification plus pancreatic ribonuclease digest (14) gave partial sequence information but still failed to distinguish between U-U-C-U-C-C-C-G and U-U-C-C-U-C-C-G. In this case, a partial venom phosphodiesterase digestion was carried out and the products were analyzed using the method set out by Ling for DNA fragments (15). The evidence obtained supported the sequence U-U-C-U-C-C-C-G but could not be called absolutely conclusive due to the very small amounts of product being analyzed at the end of the experiments. (With all the elaborate methods available for sequencing oligoribonucleotides, the sequence of a long pyrimidine chain still presents considerable difficulties.) Because of the streaking of the T1 fragment containing the 3' end (Table II, Spot 21) on a DEAE-cellulose ionophoretogram (cf. Fig. 7), it was originally felt that this species might be modified on its terminal nucleotide. Precedence for this resides with the transfer RNAs and with the finding of a blocked 3' end in one of the 4.5 S RNA molecules (4.5 S RKAIII) in mammalian cells (18). Treatment of E. coli 4.5 S RNA with aqueous ammonia before digestion, under conditions which would readily remove any acyl group, failed to change the properties of the 3' end fragment. Moreover, this fragment did not streak on polyethylenimine cellulose (Fig. 3) and was susceptible to periodate oxidation (19). Therefore, the observed streaking on DEAEcellulose was felt to be a function of the large number of cytidine residues in the fragment. The structure (I-C-C-A-C-C-CUE was assigned to the 3'.terminal fragment because it contained the same ratio of A:C (1:5) whether it was digested with TZ-ribonuclease (to nucleoside 3'.phosphates) or with venom phosphodiesterase (to nucleoside 5'.phosphates), and a fragment obtained from Uz-ribonuclease digest had an A:C ratio of 1:3.
The other difficulties encountered in fragment sequencing could be attributed to the heterogeneity of 4.5 S RNA. Working with a single strain of E. coli, the quantitations of the fragments containing A-A-G seemed to vary frorn preparation to preparation. There were also strain variations. Although the yield of A-A-G itself (obtained from T1 plus pancreatic ribonuclease A digestion), in samples from MRE 600, remained constant at 3.0 FIG. 4 (bottom right). An autoradiogram of a two-dimensional separation of the pancreatic ribonuclease A terminal digestion nroducts of 32P-labeled 4.5 S RNA from MRE 600. The techniques '-~are the same as those described in Fig. 3 except that the entire second dimensional separation was carried out in 1.6 M pyridinium formate. Missing from this photograph is cytidine 3'.phosphate (C); it runs slower than all other products in the first dimension and faster in the second. 4.5 S RNA from Escherichia coli CA 265 gave, in addition to products shown, 1 molar equivalent of A-G-G-G-C; its position is indicated. B, position of the xylene cyan01 blue dye marker.   gave GzAzC + G-G + A-A, Spot 5 gave G-G + G-U, and Spot 6 gave A-A + G-G + G-G-C.
c Indicates that CMCT blocking, followed by pancreatic ribonuclease A digestion, was used to locate the positions of U in the fragments.
Products of digestion were separated by onedimensional electrophoresis on Whatman 3MM paper, at pH 3.5. CMCT-modified products migrated toward the cathode and are given a posit,ive sign relative to the origin; unmodified products migrated toward the anode and are given a negative sign. Product identity (after removal of the blocking group) is given in parentheses. Migration of products were : Spot 1 at t-12.0 (U-U-U-A-C), $0.5 (A-G) and -3.5 cm (C); Spot 2 at +5.0 (U-U-C), +2.5 (U-C), -3.5 (C), and +3.5 and -7.0 cm (G); Spot 5 and the pentanucleotide from Uz-ribonuclease digest of Spot 3 at t-11.0 and +5.3 (U-G), and -3.5 cm (C); Spot 12 at +2.5 (U-C), -3.5 (C) and +3.5 and -7.0 cm (G and Spots 2 and 6 in pancreatic A digest) as they represent averages from several preparations.
The determination of the sequences of pancreatic ribonuclease A fragments (Fig. 4) G3A2C and G2A2C was complicated by the heterogeneity present in individual samples of 4.5 S RNA. Analysis of a partial spleen phosphodiesterase digest of both fragments using the two-dimensional procedure of Ling (15) showed that G2A2C appeared to have the sequence A-A-G-G-C regardless of its molar yield; in some species of E. coli there was only 1 molar equivalent, but in most it was nearer 2. There was, however, some indication of ambiguity in GaA#Z, and in E. coli CA 265 some evidence of a species G.,A&. The sequence given, G-G-A-A-G-C, can only be said to represent the major species with this composition in MRE 600. Moreover, in E. coli CA 265, a base change (A + G) was found in the stem portion of the molecule, which resulted in the appearance of A-G-G-G-C (rather than A-A-G-G-C) among the pancreatic ribonuclease A digests and C-A-G among the T1 products. The results obtained on analysis of the pancreatic A complete digestion products of 4.5 S RNA arc summarized in Fig. 4 and Table II. The heterogeneity of 4.5 S RNA will be considered in more detail below.

Fragment Overlaps in Sequence
Determination-In an attempt to get large oligoribonucleotides for fragment overlaps, enzymatic digestions at low temperature, with low concentrations of either 'I'1 or pancreatic ribonuclease A, were carried out. Analysis of the products obtained under a variety of conditions, using the homochromatography method (20) showed that 4.5 S RNA, unlike most other low molecular weight RNAs studied, gave mainly extremely large fragments (not separable by the method used) or products expected from complete enzymatic digestion. There were a few molecules of intermediate size, but in very low concentrations.
The identity of molecules obtained by this method is shown schematically in Fig. 10. It seemed unlikely that this technique could be made to yield a complete sequence for 4.5 S RNA.
When partial digests with T,-ribonuclease, however, were examined by electrophoresis on acrylamide gels, a series of discrete bands was observed, as shown in Fig. 5. The slowest migrating band (A) was found to give a fingerprint corresponding to that of whole 4.5 S RNA. The next band (B) gave a similar T1 fingerprint, but with two unique products missing: U-C-C-G and A-A-G. The most notable feature of Band B was that it contained both the 3' and 5' end products. Continued analysis of other bands (C to If') showed this phenomenon to persist: the fingerprints became increasingly simpler as the bands decreased in size, but each band contained the 3'. and 5'-terminal fragments. This suggested that the secondary structure, possibly in conjunction with tertiary structure, left only one area (or a very few areas) open to enzymatic attack, and further digestion proceeded from this area (or arcas) in the directions of the termini, and that the terminal portions of the molecule possessed an unusually stable structure. A less likely assumption was that if "randomly" digested to small fragments, their secondary structures were strong enough for them to remain associated with one another in a specific manner during separation by gel electrophoresis.
For further analysis, the gel bands obtained from similar partial enzymatic digests (Fig. 5) were excised, the RNA was eluted, and the 3'-and 5'.terminal "stem strands" were separated. The separation procedure used was the two-dimensional homochromatography method of &ownlee and Sanger (20).
For the larger products (B to C'), the separation was essentially accomplished by the first dimension, but for the smaller products (D to F), both dimensions afforded separation. It was hoped that Band A would be composed of two "halves" of the entire molecule, isolation of which would reveal the site most vulnerable to enzymatic digestion; all attempts to separate Band A into two fragments were, however, unsuccessful. Fig. 6 shows the separation of the two products from gel Bands D and E. In all cases (Bands B to F), the 5'-terminal strand migrated faster than the 3'-terminal in the first dimension and in most cases slower in the second. The spots corresponding to separated strands were eluted; one-half of the RNA from each spot was digested completely with Tr-ribonuclease and the other half with pancreatic ribonuclease A, and fingerprints made to allow analysis of the separated strand fragments. Fig. 7 shows Tlribonuclease fingerprints of whole 4.5 S RNA and of products from the separated strands of Bands D and F; Fig. 8 shows pancreatic ribonuclease fingerprints of whole 4.5 S RNA and the products from separated strands of Band D. Similar fingerprints were made for separated strand products from Bands B to F obtained by partial digestion with either Tl-or Ni-ribo: nuclease, and a band (C') obtained only from the latter (Fig. 5). The findings are summarized in Tables III and IV. Two points seem worthy of note in conjunction with these results. One is that gel Band C', and the digestion pattern it represents, is peculiar to the Neurospora crassa guanosinespecific ribonuclease (NJ and is not found with Tl-ribonuclease. Pinder and Gratzer (21) found that although the primary specificity of these two enzymes is the same, the cleavage patterns of rabbit reticulocyte RNA under limiting digestion conditions with the two enzymes was different. Both results point to different subsite affinities for these enzymes. The second point to note is that the enzyme cleavage leading to the 3' strand portion in gel Band C is due to cutting in what is normally a minor 4.5 S species, one in which the cytidine at position 58 has been replaced by guanosine.
Attempts to obtain large oligonucleotides under limiting digestion conditions with pancreatic ribonuclease A were only successful under extreme conditions: in the presence of large excess of carrier RNA with minute amounts of enzyme. TWO fragments, obtained under these conditions and separated by a two-dimensional acrylamide gel electrophoretic procedure, were found to come from the "non-stem" portion of the molecule and to contain the overlaps missing from the above Ti-ribonuclease results. They are labeled 6 and 7 in Fig. 9. FIG. 5 (top). An autoradiogram of a separation by gel electro-A primary structure for 4.5 S RNA is proposed in Fig. 10, phoresis of the products from partial digestions of 32P-labeled 4.5 based on the sequences of the products obtained by complete S RNA with Tl-and N1-ribonucleases at 4". Gel conditions used were similar to those described in Fig. 1.1, digestion with N1-ribo-digestion with ribonucleases (Table II) and the overlaps obnuclease using an enzyme to substrate ratio of 1:320. 2 and 3, served by digestion under limiting conditions (Figs. 5 to 10).
digestions with Tl-ribonuclease using enzyme to substrate ratios The secondary structure (Fig. 11) attempts to account not only of 1:250 and 1:500, respectively. In similar experiments with T1 for the unusual stability of this molecule but also for the sites of using enzyme to substrate ratios of 1:750 and 1:lOOO (not shown), cleavage by T1-and Ni-ribonucleases. the gel patterns were the same but most of the material was undigested (Band A) 4.5 S RNA. Bands B to F were excised and the products were isolated and analyzed; they were all found to con-DISCUSSION tain associated fragments composed of the 5' and 3' ends of 4.5 S RNA. Results are shown in Figs. 6, 7, and 8 and are summarized An early report of an RNA molecule in E. coli intermediate in Tables III and IV. in size between 5 S and transfer RNAs was made by Hindley FIN. 6 (bottom). Autoradiograms of two-dimensional separa-in 1967, when he isolated an impure species from MRE 600 (23). tions of the associated 3' and 5' stem fragments from partially digested 32P-labeled 4.5 S RNA obtained from acrylamide gel Subsequently, a number of strains of E. coli have been ex-Bands D and E (Fig. 5). Separation in the first dimension was by amined (as indicated) and been found to contain 4.5 S RNA; electrophoresis on cellulose acetate at pH 3.5 (14) and in the it has also recently been reported present in some stringent and second by thin layer chromatography on DEAE-cellulose using relaxed strains studied by Ikemura and Dahlberg (24). Molehomochromatography (20). These procedures were also used to cules of similar size, although probably unrelated, have been separate the 5' and 3' associated stem fragments from Bands B, C, C', and F (Fig. 5). In all cases, the fragment containing the isolated from eukaryotic systems (18). 4.5 S RNA found in the 5'-terminal nucleotide (pG) migrated faster in the first dimension nucleus of Novikoff hepatoma ascites cells has been sequenced than the fragment containing the 3'-terminal nucleoside (Con), as (25). A review by Weinberg (26) discusses the low molecular illustrated by D-(6') and E-(6'), and D-3' and E-S', respectively, weight RNAs found in eukaryotic cells. B, position of the xylene cyan01 blue dye marker.
The questions posed in the study of the molecule from E. COli designated 4.5 S RNA were: where is it found in the cell, when does it appear in the cell, is it a "stable" molecule, and what is its structure and its function? In one experiment (not described herein), high salt (1 M NaCl)-washed ribosomes were separated from E. coli cell supernatant and the RNA from both fractions was isolated and analyzed by gel electrophoresis.
4.5 S RNA, together with the tRNAs, was found exclusively in the non-ribosomal, supernatant portion of the cells.
In a time course study, beginning from a resting culture of MRE 600, 4.5 S RNA was found in relatively high yield in cells isolated during both early and late log phase growth. The most notable aspects of this study was that another small RNA, called 6 S RNA and sequenced by Brownlee (a), was present in very low yields at early log phase, only appearing in "normal" yields late in the cell cycle. Using AA-157, a temperature-sensitive mutant of E. coli, Griffin and Baillie found and in part characterized a precursor of 6 S RNA (8). In the same experiments, no precursor of 4.5 S RNA was found. The experiments suggested that if 4.5 S RNA has a precursor, maturation occurs early in the cell cycle.
The E. coli mutant AA-157 shows a complete shut-off of stable RNA production within minutes of being shifted from permissive to nonpermissive temperature, whereas most messenger RNA production is unimpaired at the latter temperature (8,27). In a pulse-chase experiment, aimed at studying the stability of 4.5 S RNA, it was found that label 32P was not chased out of this molecule when cells were given excess nonradioactive phosphate and left at the nonpermissive temperature for times exceeding several cell growth cycles. Thus, 4.5 S RNA was found to be related to other low molecular weight RNAs such as 5 S, 6 S, and transfer RNAs in its stability.
Having thus defined 4.5 S RNA as a stable species which appears in the supernatant fraction of the cell early in the growth cycle, an attempt was made to sequence this molecule with the hope that its structure might shed some light on its function. Sequence analysis was complicated by the fact that several related species were found in material isolated from whole cell MRE 600. Attempts to separate these species by biological or chemical methods were without success, although purification of the RNA to a high degree led to the predominance of two FIG. 7 (top). Autoradiograms of two-dimensional separations of terminal T1 -ribonuclease digestion products of whole 4.5 S RNA from MILE 600 and the separated 5' and 3' stem fragments from gel Bands D and F (cf. Fig. 5). Separation in the first dimension was by electrophoresis on cellulose acetate strips, pH 3.5, and in the second by electrophoresis on sheets of DEAE-cellulose (43 X 85 cm, DE81) in 7y0 formic acid (14). For accurate comparison of the position of oligonucleotides, the digestion products from the separated 5' and 3' fragments of Band D, and similarly of Band F, were each put on the same strip of cellulose acetate and applied at one end and in the center (D-3' and D-5', and F-3' and F-5', respectively, as shown) electrophoresis was carried out and the products were separated further by a second dimension on the same sheet of DE81. The oligonucleotides belonging to the 3' portion of the molecule can be seen in the right half of the photographs of products from Bands D and F, and those belonging to the 5' portion of the molecule in the left half. Numbers 21 and 6 show the positions of the 3'. and 5'-terminal T1 products (C-C-C-A-C-C-Co= and pG, respectively). The identities of the other numbered products are given in Table  II. All products were isolated and identified as described in the text. B, position of the xylene cyan01 blue dye marker. Similar two-dimensional analyses (not shown) were carried out on the T1 products from the separated 3' and 5' associated fragments of gel Bands B, C, C', and E (Fig. 5). The results are summarized in Table  III. FIG. 8 (bottom).
Autoradiograms of two-dimensional separations of terminal pancreatic ribonuclease A digestion products of whole 4.5 S RNA from MRE 600 and the separated 5' and 3' stem fragments from gel Band D (Fig.  5). The procedures used were identical with those described in Fig. 7. (Uridine-3'-phosphate (Spot 16) does not appear on the photograph of the pancreatic ribonuclease A digest of whole 4.5 S RNA; it would be the fastest running spot in the second dimensional separation.) The oligonucleotides belonging to the 3' portion of the separated fragments from gel Band D can be seen in the right half of its photograph and those belonging to the 5' portion in the left half. Number 1 shows the position of the 5'-terminal product, pG-G-G-G-C. The identities of all the other numbered products are given in Table  II. B, position of the xylene cyan01 blue dye marker. Similar twodimensional analyses (not. shown) were carried out on the products from the 3' and 5' associated fragments of gel Bands B, C, C', E, and F (Fig. 5). The results are summarized in Table   IV.
FIG. 9. An autoradiogram of a two-dimensional acrylamide gel electrophoretic separation of the products from a partial pancreatic ribonuclease A digest (enzyme to substrate ratio, 1:23000) of 4.5 S RNA.
The conditions for the first dimensional separation were the same as those given in Fig. 1. The gel band (2 cm wide) containing the separated products was soaked in 7 M urea, then applied horizontally to the top of a 12.5%-acrylamide stacking gel (20 X 20 cm) essentially as described by Vigne and Jordan (13) and electrophoresis was carried out in borate buffer, pH 8.3. Spots 1 to 7 were eluted, further digested with pancreatic ribonuclease A, and analyzed by the two-dimensional methods described in Figs. 7 and 8. The results on Spot 1 showed it to contain species differing by only a single C + G base change. A corollary of this finding is that there must be at least two gene copies, and possibly more, for 4.5 S RNA. The products of complete digestion of 4.5 S RNA with either Ti or pancreatic A ribonucleases were examined (Figs. 3 and 4), The characterization of each product of complete digestion was determined using methods previously described (cf. Table II).
Sequence analysis was complicated by what in hindsight can be seen as the secondary structure of the molecule. Under limiting enzymatic digestion conditions, it proved to be difficult to get oligonucleotides of intermediate size, necessary for determining the overlaps of the products of complete enzymatic digestion.
Most digestion conditions gave either the latter products themselves, or products of size too large for analysis on the usual two-dimensional systems (14). Oligonucleotides of intermediate size, some of which were isolated and partly characterized, were generally present in yields too small to allow for absolute identity determination.
This problem was solved when the affinity for association of stem portions of the molecules was recognized. 4.5 S RNA was digested under limiting digestion conditions with Ti-ribonuclease and the associated stem fragments were separated by electrophoresis on acrylamide gels (cf. Fig. 5). The isolated fragments were then further separated into 3'-and 5'terminal species under denaturing conditions and each fragment subsequently was digested and analyzed. The largest associated stem fragment contained 98 of the products belonging mainly to the 3' end of 4.5 S RNA (Table  IV) whereas those from Spot !J belonged mainly to the 5' end of the molecule.
They were not, however, fully characterized.
Spots 3 to 5 were not pure enough for accurate characterization but contained products expected from associated 3' and 5' stem fragments.
Spots 6 and 7 were found to contain products belonging to the center of the molecule and were fully characterized by the two-dimensional procedure shown in Figs. 7 and 8. Spot 6 contained sequences corresponding to a region of the molecule extending from position 52 to 87, and Spot 7 from position 41 to 58 (Fig. 11) The letters A to E indicate main cleavage sites using partial digestion conditions with T1-ribonuclease and correspond to letters used in Fig. 5. Numbers 6 and 7 show sites of cleavage obtained under partial pancreatic ribonuclease A digestion conditions and correspond to numbers shown in Fig. 9. The secondary structure suggested takes into account thermodynamic considerations (28), the general behavior of the molecule, and the sites susceptible to enzymatic cleavage. This structure should be more stable than that suggested previously (22).