[The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it]. / K voprosu o sushchestvovanii v skeletnykh myshtsakh krolika vetviashchego fermenta ribonukleoproteidnoi prirody i sootvetstvuiushchego ribozima.

Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.

[Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods]. / Temperaturozavisimye strukturnye izmeneniia v kompleksakh tRNK(adenin-1)-metiltransferazy iz Thermus thermophilus HB8 i tRNK, issledovannye opticheskimi i élektronno-mikroskopicheskimi metodami.

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.

[A study of tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8 with tRNA using enzymatic and spectral methods]. / Izuchenie kompleksov tRNK(adenin-1-)-metiltransferazy iz Thermus thermophilus HB8 s tRNK fermentativnymi i spektral'nymi metodami.

The contact sites of yeast tRNA1Val with tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) were studied by comparing the partial digests of free and enzyme bound tRNA. The RNAases Sa, V1, S1 and A were used for this purpose. Phosphodiester bonds on the proximal end of the acceptor stem and adjacent D- and T psi-stems, forming a continuous area, are protected by the methylase from the action of RNAases. On the contrary an enhancement of phosphodiester bond splitting of the D- and T psi-loops and of the anticodon stem is observed in the presence of methylase, which is interpreted as tertiary structure relaxation of tRNA owing to complex formation. The isotherm of ethidium bromide adsorption on free and methylase bound tRNA has shown that in enzyme shields approximately 50% of tRNA double stranded regions.

[Interaction of s4U8 region of tRNA Phe with tRNA-(adenine-1-)-methyltransferase from Thermus thermophilus]. / Vzaimodeistvie raiona s4U8 tRNK Phe s tRNK-(adenin-1-)-metil- transferazoi iz Thermus thermophilus.

Photoaffinity labelling of tRNA (adenine-1-)-methyltransferase with an E. coli tRNA(Phe) derivative bearing 4-azidophenylmercuro group attached to s4U residue as well as direct photocross-linking of the native tRNA(Phe) with the enzyme via s4U residue has been studied. Both techniques labelling gave similar results, leading to covalent attachment of tRNA(Phe) to the enzyme within a specific complex. The data obtained indicate unambigously that s4U residue contacts with tRNA (adenine-1-)-methyltransferase within the corresponding specific complex.

[RNA M1 and other catalytically active polyribonucleotides]. / RNK M1 i drugie kataliticheski aktivnye poliribonukleotidy.

A review of data on the RNA components of RNAase P which proved to be the catalytic subunits of the enzyme is given. The properties of the polyribonucleotides, the comparison of their structure and the principles of their action, suggested at the time being, are discussed.

Catalytic activity of the nucleic acid component of the 1,4-alpha-glucan branching enzyme from rabbit muscles.

2.5 S RNA, the nucleic acid component of the 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase from rabbit muscles, devoid of any protein, catalyses the branching reaction, as does the holoenzyme. The conclusion is drawn that 2.5 S RNA is a ribozyme. To get an insight into the significance of different parts of the molecule for the catalytic activity of 2.5 S RNA, a large fragment isolated from its partial RNAase A digest was investigated. This fragment which proved to be the middle part of polyribonucleotide chain containing all modified nucleotides exerts some catalytic activity, too.

[tRNA genes of pro- and eukaryotes. II. Gene expression and processing of gene products]. / Geny tRNK pro- i éukariot. II. Ekspressiia genov i protsessing gennykh produktov.

The up-to-date state of investigations on cytoplasmic tRNA genes expression and processing of gene products are reviewed. Special emphasize is given to data on RNAase P and splicing.

[The tRNA genes of pro- and eukaryotes. Organization, structure, transcription]. / Geny tRNK pro- i éukariot. 1. Organizatsiia, struktura, transkriptsiia.

The up-to-date data on cytoplasmic tRNA genes, their number and structure, as well as organization in the genome and transcription are reviewed.

[tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8]. / tRNA(adenin-1-)-metiltransferaza iz Thermus thermophilus HB8.

tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) was isolated from the extreme thermophile Thermus thermophilus strain HB8. The specific activity of the enzyme is about 50 000 and the yield of activity more than 20%. The method of isolation consists of five steps and is valid for isolation of mg quantities of the enzyme. The purified protein preparation is practically homogeneous in SDS-gel electrophoresis, the position of the protein band corresponds to a molecular weight of 25 000. By gel filtration on Sephadex G-100 the molecular weight of the native protein was found to be 70 000. These data allow to suggest a subunit structure of the enzyme. The enzyme is highly thermostable and is most active at 80 degrees C. The only activity of the enzyme is to methylate A58 in the T psi X loop of tRNA.

[Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide]. / Izuchenie 2,5S RNK 1,4-alpha-gliukanvetviashchego fermenta fluorestsentnymi metodami s ispol'zovaniem bromida étidiia.

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.

Purification and characterization of tRNA (adenine-1-)-methyltransferase from Thermus flavus strain 71.

tRNA (adenine-1-)-methyltransferase was isolated from the extreme thermophile Thermus flavus, strain 71. It was purified about 2000-fold by ammonium sulfate fractionation and affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3' end. The purified protein preparation is free of nuclease and aminoacyl-tRNA synthetase activity and contains no more than 4% of tRNA (guanine-7-)methyltransferase activity. The only activity of the enzyme is to methylate A58 in the T psi loop of tRNA. Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as a substrate. The enzyme is highly thermostable. It is most active at 75 degrees C. tRNA (adenine-1-)-methyltransferase has a Km of 0.4-0.5 microM for tRNA2Gln from Escherichia coli and a Km of 6 microM for S-adenosyl-L-methionine.

[Processing of tRNA]. / Protesessing tRNK.

The article reviews various aspects of tRNA biosynthesis in pro- and eukaryotes. Some data on the tRNA gene localization are presented. Structures of tRNA precursors are given; their conversion into mature tRNA molecules and the corresponding enzymatic systems are discussed in detail. Special emphasis is given to the transcription of tRNA genes and processing of tRNA precursors in vitro. Similarities and diversities of tRNA processing in pro- and eukaryotes are discussed.

Primary structure of the nucleic acid from the 1,4-alpha-glucan branching enzyme.

The primary structure of the nucleic acid from the branching enzyme 1,4-alpha-D-glucan: 1,4-alpha-D-glucan 6-alpha-(1,4-alpha-glucano)-transferase (2.5-S RNA) isolated from rabbit muscles has been elucidated. The polyribonucleotide consists of 31 nucleotides; the unique features of the polyribonucleotide are the unusually high content of modified nucleotides (32%) and guanine residues (40%). Apparently 2.5-S RNA belongs to a class of nucleic acids unknown up to now. It is the first time that the structure of a nucleic acid component from a ribonucleoenzyme has been defined. This work is a preprequisite for gaining insight into the intimate activating effect of the poly-ribonucleotide on the enzyme action.

[tRNA-methylase study of the extreme thermophile, Thermus flavus]. / Izuchenie tRNK-metilaz ékstremal'nogo termofila Thermus flavus.

tRNA methylases were studied in the extreme thermophilic culture of Thermus flavus, strain 71. Like E. coli, the culture contained only those tRNA methylases which catalysed the formation of m1A and m7G. Mg2+, Ca2+ and Na+ ions activated tRNA methylases of Thermus flavus in the series Mg greater than Ca greater than Na while Mn2+ ions inhibited the enzyme. The activity of tRNA methylases was higher in T. flavus than in E. coli, and required less protein and time for exhaustive methylation of tRNA preparations. The overall activity of methylases in T. flavus at 70 degrees C was 5-6 times higher than at 40 degrees C; the elevation of temperature had different effect on various methylases: the activity of m1A methylase increased 13-fold whereas that of m7G methylase only twofold.

The mechanism of action of tRNA methylases studied with immobilized tRNAs.

Each of the individual tRNAs immobilized on aminohydroxybutyl-cellulose (ABC) through their oxidized 3'-terminal binds affinitively all methylases present in the enzyme extract irrespective of whether this tRNA will be involved in the following step of methylation or not. These data allow to suggest that (a) the formation of a methylase-tRNA complex and the catalytic act of methylation are indeed autonomous processes and (b) the first step of interaction between tRNAs and tRNA methylases is rather unspecific and consists in the recognition of the whole class of tRNA molecules.

[Amino oxyadsorbents. New type of adsorbents for affinity chromatography: purification of tRNA-methylases from rat nephron on aminooxybutylcellulose with immobilized tRNA]. / Aminooksiadsorbenty. 8. Novyi tip sorbentov dlia affinnoi khromatografii: ochistka tRNK-metilaz nefromy krysy na aminooksibytiltselliuloze s immobilizovannoi tRNK.

A new type of sorbents for affinity chromatography is suggested and used to purify tRNA methylases. tRNA was immobilized on aminooxybutylcellulose via the oxidized 3'-end. In order to bind other enzymes specific for nucleic acids in general, e. g. nucleases, and to achieve a higher degree of purification the crude enzyme preparation was treated with rRNA immobilized on aminooxybutycellulose. The sequential application of two sorbents mentioned allows to get an approximately two hundred fold purification of total tRNA methylases. In a separate experiment the possibility of individual tRNA methylase fractionation by means of elution with a NACl gradient was shown; the degree of purification for some methylases was more than a thousand fold.

[Comparative study of the tRNA-methylases of normal and tumor tissues. I. Spectrum of renal and carcinoma RA methylases]. / Sravnitel'noe izuchenie tRNK-metilaz opukholevykh i normal'nykh tkanei. I. Sprectr metilaz pochki i kartsinomy PA.

A comparative study of rat kidney and carcinoma RA tRNA-methylase activity has been carried out using partially purified enzyme preparations and total E. coli tRNA. Also the nuclease activity of the methylase preparations from kidney and carcinoma was compared. It was established that the methylase activity in carcinoma preparations is higher, whereas the nuclease activity is lower in comparison to the enzyme preparations from liver. No formation of some specific methylated compounds could be established in the case of carcinoma. It was established that the relative contribution of individual methylases to the elevated level of total tRNA-methylase activity in carcinoma is different. Maximal enhancement of activity was established for the methylase forming m5U, whereas the activity of the enzymes, transfering the methyl group to the fifth position of C is practically equal in kidney and carcinoma tissues. Experimental results and theoretical evaluation of the hypotheses suggested to explain the higher methylase activity in tumor tissues allowed to reject some of them.