Showing papers in "Nucleic Acids Research in 1974"

Internal structure of the chromatin subunit.

Abstract: The digestion of chromatin insitu with DNase I reveals, after denaturation, a regular series of single stranded DNA fragments the lengths of which represent multiples of 10 bases. These experiments are compatible with the DNA being on the outside of the chromatin subunit and suggest that the subunit structure itself contains repetitive structural elements. Possible models are discussed.

Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ.

Abstract: A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of (14)C leucine incorporated into protein in a 50 mul assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg(+2), K(+) and spermine concentration as well as the method of extract preparation. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.

DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping

Abstract: Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresisdagger; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and seven from the right-hand 3'-terminus of bacteriophage lambda DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and five nucleotides from the right-hand terminus of bacteriophage phi80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

A model for particulate structure in chromatin

Abstract: A model is proposed for the structure of nuclease-resistant chromatin particles. The model is novel in that it proposes that the DNA in such a particle is wound about a protein core, made up of the hydrophobic regions of histone molecules.

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12.

Abstract: Four hitherto undescribed endodeoxyribonucleases, temporarily designated A(1), A(2), A(3), and B, have been isolated from E. coli K-12. Each requires Mg(++) and is not stimulated by ATP or S-adenosylmethionine. A(3) is strongly inhibited by Fe(+++) and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3'-phosphatase. A(1) (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3'-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A(2) (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3'-hydroxyl- and 5'-phosphoryl termini. A(3) (molecular weight approximately 38,000) cleaves fd DNA to form 3'-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A(3). B appears to form 3'-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.

“Methylation-coupled” transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA

Abstract: S-adenosyl-L-methionine (SAM) activated the virus-associated RNA polymerase of cytoplasmic polyhedrosis virus in vitro. Synthesis of single-stranded viral RNA (mRNA) proceeded depending on the presence of SAM.A methyl residue of SAM was incorporated into an RNA molecule. A ribose moiety of adenylic acid in the 5'-terminal region of the nascent RNA was methylated in the very early stage of the transcription. The dependence of the viral transcription on the presence of SAM and the methylation of terminal nucleotide suggests that the transcription of CPV is a "methylation-coupled" reaction.

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

Abstract: The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) . poly (dT), poly (dA-dT) . poly(dA-dT) and poly (dI-dC) . poly (dI-dC) but poorly, if at all, to poly (dG) . poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K(a)=2.9 . 10(5) M(-1)); corresponding values for distamycin A are one site per 6.1 nucleotides with K(a)= 11.6 . 10(5) M(-1). Binding sites apparently involve predominantly A.T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3 degrees per bound antibiotic molecule.

Temperature sensitive mutants of Escherichia coli for tRNA synthesis

Abstract: An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3(+)-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA(Tyr) was demonstrated. Electrophoretic fractionation of (32)P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions. In spite of much information on the structure and function of transfer RNA (tRNA), our knowledge concerning the biosynthesis of tRNA is relatively poor. It is generally assumed that complete tRNA molecules are made via a series of processing steps from the original transcription products of tRNA genes which are presumably unmodified and longer than mature tRNA molecules. In the case of tyrosine suppressor tRNA of su3(+), an unmodified precursor RNA carrying additional residues at the 3' and 5' ends has been isolated (1,2), and an endonuclease cleaving at the 5' side of this precursor has been identified in E. coli (3). In the case of T4 encoded tRNA, a large precursor molecule for several tRNA's has been reported (4). Some enzymes that catalyze the modifications have also been described (5). However, the over-all picture and the precise mechanisms of tRNA maturation are as yet largely unkown. For study of tRNA biosynthesis in E. coli, a genetic approach may prove useful, as has been the case in other biosynthetic pathways. In order to obtain mutants blocked in any of the intermediary steps of tRNA synthesis, we have developed an efficient selection system that enriches these mutants. Since any mutational block in tRNA biosynthesis might well be lethal, we looked for conditional lethal mutants in which the defect in tRNA synthesis occurs only at high temperature. In this selection system, the su3 gene carried by a temperate phage was newly introduced into cells(su(-)) and those cells incapable of synthesizing su3(+) tRNA at high temperature were selected. Such mutants were easily enriched by using conditions in which cells expressing suppressor activity were killed by two virulent phages. In this communication, we report the method for isolation of mutants and some characterization of tRNA synthesis in these mutants. Recently, Schedl and Primakoff (6) have independently isolated thermosensitive mutants of E. coli defective in tRNA synthesis which may or may not be different types from ours.

Studies on transfer ribonucleic acids and related compounds. IX(1) Ribo-oligonucleotide synthesis using a photosensitive o-nitrobenzyl protection at the 2′-hydroxyl group

Abstract: o-Nitrobenzyl group was introduced to the 2'-hydroxyl function of uridine via 2',3'-O-(dibutylstannylene) uridine. The benzylated uridine was protected at the 5'-hydroxyl group with monomethoxytrityl chloride and condensed with 2',3'-O-dibenzoyluridine 5'-phosphate or N,N',2',3'-O-tetrabenzoyladenosine 5'-phosphate using dicyclohexylcarbodiimide (DCC). o-Nitrobenzyl ether linkage of the dinucleotides was removed by UV irradiation with wavelength longer than 320 nm. Deprotected UpU and UpA thus obtained were characterized by RNase A digestion.

The mapping and ordering of fragments of SV40 DNA produced by restriction endonucleases

Abstract: SV40 DNA is cleaved by the Eco RII and Hae restriction endonucleases to give rise to two different sets of 16 fragments each. These fragments have been ordered by analysis of the products of redigestion of one set of fragments with another restriction enzyme. The cleavage sites have been precisely mapped based on length measurements of the products of each cleavage. This provides a convenient group of small DNA fragments suitable for sequence analysis investigation of the transcripts present in infected cells, or construction of deletion substitution variants of the virus.

The nucleotide sequence preceding an RNA polymerase initiation site on SV40 DNA. Part 1. The sequence of the late strand transcript

Abstract: The nucleotide sequence of the RNA transcript from the “early” (E) strand of SV40 DNA immediately preceding the preferred E. coli RNA polymerase start site is G-(A-A-A-C, -A-U-)-A-A-A-A-U-G-A-A-U-G-C-A-A-U-U-G-U-U-G-U-U-G-U-U-A-A-C-U-U-G-U-U-U-A-U-U-G-C-A-G-C-U-U-A-U-A-A-U-G-G-U-U-A-C-Ap. The last nucleotide of the sequence is the first nucleotide transcribed by E. coli RNA polymerase from the “E” strand. The DNA template contains a palindrome of 17 residues that includes the Hemophilus influenza restriction endonuclease cleavage site G-T-T-A-A-Cp. The DNA which gives this transcript lies very close to one end of SV40 DNA segment in the Adeno-SV40 hybrid virus Ad2+ND3 and appears to contain sufficient untranscribed information to specify the E. coli RNA polymerase start.

Correlation between three-dimensional structure and chemical reactivity of transfer RNA.

Abstract: The bases of yeast tRNAPhe which react with carbodiimide and methoxyamine have been determined and this information has been combined with chemical modification studies of other workers to produce a composite picture of base accessibility in this tRNA. The results are compared with the three-dimensional structure which we have recently determined. The bases which react chemically lie in exposed positions in the three-dimensional model and those which do not are either in the double helical stem regions or else are involved in maintaining the tertiary structure through pairing or stacking interactions.

Poly(adenosine diphosphate-ribose) polymerase in quail oviduct. Changes during estrogen and progesterone induction.

Abstract: The activities of the following enzymes have been determined in nuclei of quail oviducts in response to exogenous stimulation of the birds with diethylstilbestrol, used as an estrogen analogue and progesterone: DNA dependent DNA polymerase, DNA dependent RNA polymerase I and II and poly(adenosine diphosphate-ribose) [=poly(ADP-Rib)] polymerase. During primary stimulation with the estrogen analogue the activities of the four DNA dependent polymerases increase to about the same degree. Upon withdrawal of the hormones the levels of the enzymes drop to values known from nuclei from unstimulated quail oviducts. The secondary stimulation with the estrogen analogue causes a significant increase only of the RNA polymerase II. The in vivo induction of avidin by progesterone in oviduct mucosa cells from quails, during the period of primary estrogen stimulation, is accompanied by an increase of RNA polymerase II activity and a marked decrease of poly(ADP-Rib) polymerase activity. The activities of RNA polymerase I and of poly(ADP-Rib) polymerase are not affected significantly by an exogenous administration of progesterone.

Repeated DNA sequences in fungi

Abstract: Several fungal species, representatives of all broad groups like basidiomycetes, ascomycetes and phycomycetes, were examined for the nature of repeated DNA sequences by DNA:DNA reassociation studies using hydroxyapatite chromatography. All of the fungal species tested contained 10-20% repeated DNA sequences. There are approximately 100-110 copies of repeated DNA sequences of approximately 4 × 107 daltons piece size of each. Repeated DNA sequence homoduplexes showed on average 5°C difference of Te50 (temperature at which 50% duplexes dissociate) values from the corresponding homoduplexes of unfractionated whole DNA. It is suggested that a part of repetitive sequences in fungi constitutes mitochondrial DNA and a part of it constitutes nuclear DNA.

New fluorescent hydrazide reagents for the oxidized 3'-terminus of RNA

Abstract: The synthesis and properties of four new fluorescent reagents capable of forming moderately stable links to the 3′ oxidized end of RNA are reported. All are hydrazide derivatives: pyrene butyric acid hydrazide, proflavine monosemicarbazide, proflavine monosuccinic acid hydrazide, and anthracene-9-carboxaldehyde carbohydrazone. In addition, procedures are given for coupling the bifunctional reagent carbohydrazide to the 3′ end of RNA. These carbohydrazide adducts can easily be coupled in turn to a wide variety of fluorescent reagents having specificity for aliphatic amino groups, including isothiocyanates and sulfonyl halides. Thus a route exists for the preparation of an enormous variety of 3′ fluorescent labeled RNAs. The carbohyrazide adducts are also useful for other synthetic procedures such as preparation of covalent tRNA dimers.

Isolation and sequence determination of the 3′-terminal regions of isotopically labelled RNA molecules

Abstract: The method which was developed for the selective isolation of 3'-terminal polynucleotides from large RNA molecules on columns of cellulose derivatives containing covalently bound dihydroxyboryl groups has been modified and adapted for use on radioactively labelled RNAs. The 3'-terminal polynucleotide fragments which result from specific ribonuclease digestion of isotopically detectable quantities of RNA can be selectively obtained in both high yield and purity by the modified procedure and can be subsequently analyzed by standard electrophoretic and chromatographic techniques. In addition, when the extent of enzymatic fragmentation of the RNA is controlled, the procedure permits the selective isolation of discrete "sets" of fragments of variable chain length, all of which derive from the 3'-terminus of the RNA molecule. These overlapping polynucleotides can be used directly to obtain extensive sequence information regarding the primary structure in the 3'-region of the RNA.

Thermodynamic and kinetic properties of short RNA helices: the oligomer sequence AnGCUn

Abstract: We studied the thermodynamic, kinetic and optical properties of the double helices formed by the series of self-complementary oligonucleotides, (AP)nGpC(pU)n, 2 ≤ n ≤ 4, and found that the shortest helix, containing just 6 base pairs, is less stable than would be predicted from the properties of the larger molecules. It also shows a markedly smaller hyperchromism on melting than expected. These anomalous properties of a short helix indicate that one cannot always assume that base pair free energies and extinction coefficient changes are independent of helix size.

Fractionation and characterization of satellite DNAs of the kangaroo rat (Dipodomys ordii)

Abstract: Nuclear DNA from liver cells of the kangaroo rat species Dipodomysordii was fractionated and characterized with the aid of buoyant density gradients in neutral and alkaline CsCl and in Ag(+)-Cs(2)SO(4). More than one-half of the DNA was present in three density satellites, a greater proportion than in any other species yet reported; the purified satellite DNAs were denser than principal DNA. All satellite fractions revealed sharp isopycnic bands and narrow denaturation profiles. Two had identical buoyant densities but differed substantially in T(m), base composition, and reassociation kinetics. In alkaline CsCl all three satellites, as well as a shoulder of intermediate repetitive DNA on the heavy side of the principal band, revealed unique strand densities. The most highly repetitive satellite was unusually rich in (G + C) and contained 6.7% of 5-methylcytosine. A survey of internal organs and spermatozoa of an adult male revealed no significant differences in distribution of the satellites among tissues.

A new simpler photoaffinity analogue of peptidyl tRNA

Abstract: The synthesis of the n-hydroxysuccinimide ester of N-(2-nitro-4-azidophenyl)glycine (NAG) is described. This reacts with E. coli phe-tRNAPhe to yield the photoaffinity label NAG-Phe-tRNAPhe. This peptidyl tRNA analogue binds correctly to the peptidyl site of the E. coli ribosome. The only significant covalent products found after irradiation of a peptidyl site bound NAG-Phe-tRNAPhe-70S-poly(U) complex are 50S proteins L11 and L18. After irradiation the complex can still bind [3H]Phe-tRNA to the amino acyl site and participate in peptide bond formation with the covalently attached NAG-Phe moiety. Alternatively, one can allow peptide bond formation to occur first, prior to photolysis. The reaction products are still L11 and L18. Hence, both of these two proteins appear to be centrally located at the peptidyl transferase center.

Aliphatic analogues of nucleotides: synthesis and affinity towards nucleases

Abstract: DL-1-(2,3-Dihydroxypropyl)thymine was prepared by Hilbert-Johnson reaction of 2,4-dinethoxy-5-methylpyrimidine with allyl bromide followed by the osmium tetroxide catalyzed hydroxylation of the l-allyl-4-methoxy-5-methylpyrimidin-2-one obtained as an intermediate. The D-glycero enantiomer, R-1-(2,3-dihydroxypropyl)thymine and the corresponding 1-substituted uracil derivative were prepared from 3-O-p-toluenesulfonyl-1, 2-O-isopropylidene-D-glycerine and sodium salt of 4-methoxy-5-methylpyrimidin-2-one or 4-methoxypyrimidin-2-one followed by treatment with hydrogen chloride in ethanol. The phosphorylation of the above 2,3-dihydroxypropyl derivatives with phosphoryl chloride in triethyl phosphate afforded the corresponding 3-phosphates which were transformed into the 2′,3′-cyclic phosphates by the condensation with N,N′-dicyclohexylcarbodiimide. The latter compounds of the D-glycero configuration are split by some microbial RNases to the 3-phosphates.

Control of transcription of the reovirus genome

Abstract: The ten double-stranded RNA segments of the reovirus genome are not transcribed at equal frequencies until later times during the course of infection. When an inhibitor of protein synthesis such as cycloheximide was added to infected cells at the beginning of infection, only four of the ten segments were transcribed. By two hr post infection, five and possibly seven of the segments were being transcribed. By four hr post infection all ten segments were being transcribed but not yet at equal relative frequencies. The transcription pattern at intermediate (4 hr) and late (10 hr) times were verified without using cycloheximide. A repressor present in the host cell may be responsible for controlling transcription of the reovirus genome.

The nucleotide sequence of phenylalanine tRNA from Mycoplasma sp. (Kid)

Abstract: Phenylalanine tRNA from Mycoplasma sp. (Kid) was purified and characterized. The tRNA can be aminoacylated by phenylalanyl-tRNA synthetase from both Mycoplasma and E. coli. In a tRNA-dependent cell-free E. coli amino acid incorporating system programmed with poly U pure Mycoplasma tRNAPhe was fully active in promoting phenylalanine incorporation, even in direct competition with homologous E. coli tRNAPhe. Since the Mycoplasma tRNA lacks isopentenyladenosine, or any related hypermodified nucleoside, it appears that the presence of such nucleosides in tRNA is not an absolute requirement for protein synthesis.

Sequence analysis of nonradioactive RNA fragments by periodate-phosphatase digestion and chemical tritium labeling: characterization of large oligonucleotides and oligonucleotides containing modified nucleosides

Abstract: A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide dialdehydes generated by treatment of polyribonucleotides with alkaline phosphatase and excess periodate at pH 8 (borate buffer; no primary amine present in the reaction mixture). While neither phosphatase nor periodate possess any intrinsic exonuclease activity their combination mimics an RNA-specific exonuclease ("pseudo-exonuclease"). Procedures are described for separation and characterization of tritiumlabeled oligonucleotide derivatives. The sequence is deduced by identification of labeled 3'-termini following separation of the reduced nucleotide intermediates according to chain length. The sensitivity of the method is indicated by the fact that as little as 0.01 O.D.260 unit of a nonradioactive decanucleotide is sufficient for sequence determination.

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

Abstract: In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.

Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Properties of the enzyme

Abstract: A modified purification is described for an enzyme, from Escherichiacoli B, which polymerizes deoxyribonucleoside-5' diphosphates. Under appropriate conditions, the enzyme will add a single deoxyribonucleotide residue to a deoxyribo-oligonucleotide primer. At all stages, the enzyme activity copurified with the activity which will polymerize adenosine-5' diphosphate (polynucleotide phosphorylase). Studies of heat stability, the effect of various temperatures of reaction and of disc gel electrophoresis failed to provide evidence that the two activities are separable.

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia.

Abstract: The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 109 daltons for mu particle DNA and 0.81 × 109 daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 109 and 5.05 × 109 daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.

2′-O-Methyl ribothymidine: a component of rabbit liver lysine transfer RNA

Abstract: One of the lysine transfer RNAs of rabbit liver is shown to contain 2′-O-methyl ribothymidine in place of ribothymidine. This represents the first demonstration of the presence of 2′-O-methyl ribothymidine in a nucleic acid.

Synthesis of 1,N6-etheno-2-aza-adenosine (2-aza-ε-adenosine): a new cytotoxic fluorescent nucleoside

Abstract: 1,N(6)-Etheno-2-aza-adenosine was synthesized by treating 1,N(6)-etheno-adenosine with alkali, followed by nitrosation. The mechanism of formation of this novel nucleoside was elucidated using adenosine tritiated at C-8 and C-2, and was found to deformylate exclusively at C-2. This new 2-aza nucleoside fluoresces at 494 nm when excited at 358 nm. Toxicity study showed the compound is active in a rat mammary tumor tissue culture line, but inactive in HeLa and Glioma 26 tissue culture lines. It was also found to selectively inhibit the thymidine incorporation into DNA in a rat mammary tumor, but exhibits no ill effect on normal proliferative tissue. The reactive intermediate 3-beta-D-ribofuranosyl-4-amino-5-(imidazol-2-yl) imidazole was identified and was found to be an active agent in tissue culture.

Abnormal maturation of precursor 16S RNA in a ribosomal assembly defective mutant of E.coli

Abstract: The precursor and mature 16S ribosomal RNAs from a novel thermosensitive ribosomal assembly defective mutant of E. coli, in which genetic evidence suggests that the ribosomal protein S4 is altered, have been isolated and characterised by finger-printing methods. The precursor 16S RNA, which is accumulated at 42 degrees , appears to be identical with that present in wild-type strains, and with that previously described by other workers. However, the mature 16S RNA, which is contained in apparently normal functional 30S ribosomal particles synthesised at the growth-permissive temperature of 30 degrees , is incompletely trimmed and has either one or two additional nucleotides at its 5'-terminus. This might be due either to an accumulation of two late intermediates in the maturation process, or to mis-trimming of the RNA. Both possibilities suggest that the change in the protein S4 is not only responsible for the thermosensitive character of ribosomal assembly in this mutant, but also causes an alteration in the trimming site, affecting its recognition by the enzyme involved in the maturation.