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Extensions of the known sequences at the 3' and 5' ends of 23S ribosomal RNA from Escherichia coli, possible base pairing between these 23S RNA regions and 16S ribosomal RNA.

Extensions of the known sequences at both 3' and 5' ends of 23S ribosomal RNA are presented: The 5' terminal is pG-G-U-U-A-A-G-Cp or pG-G-U... G-U-U-A-A-G-Cp, with a very short sequence between Up and Gp and the 3'terminal is G-A-A-C-C-G-A-(G)-G-C-U-U-A-A-C-C-U-UOH. These two terminal regions exhibit a high degree of complementarity. In addition, extensive complementarities are also found between the 5'terminal sequence of 23S RNA and a sequence contained in section A of the 16S ribosomal RNA, and between the 3'terminal sequence of 23S RNA and sequences in sections O and J in the 16S RNA. The degree of complementarity between the two extremities of 23S RNA, and between these extremities and regions of the 16S RNA, is far greater than would be expected on a random basis suggesting a possible involvement of this base-pairing in the functioning of ribosomes. This possibility is discussed.

Base Sequence

Sequence studies on 16S ribosomal RNA from a blue-green alga.

The 16S ribosomal RNA of the blue green alga Anacystis nidulans has been characterized in terms of the oligomers generated by digestion with T1 ribonuclease. A. nidulans by this criterion is definitely a procaryote; being no more distant from Bacilli or Enterics than the latter two are from one another. A. nidulans appears to be somewhat more closely related to the Bacilli than to the Enterics.

Bacillus

Application of a rapid gel method to the sequencing of fragments of 16S ribosomal RNA from Escherichia coli.

A gel sequencing method has been applied to two 5' end-labelled fragments of the 16S ribosomal RNA from E. coli. The procedure involves partial enzymatic hydrolysis by ribonucleases T1, U2 or A, in order to generate series of end-labelled subfragments terminating in guanine, adenine, or pyrimidine residues, respectively. The two fragments concerned were approximately 75 and 90 nucleotides in length, and both arose from the 3' region of the 16S RNA. The sequences deduced are compared with the published sequence of 16S RNA, and contribute information to the final ordering of the ribonuclease T1 oligonucleotides in the latter, as well as revealing some probable errors.

Base Sequence

Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.

The complete nucleotide sequence of the 16S RNA gene from the rrnB cistron of Escherichia coli has been determined by using three rapid DNA sequencing methods. Nearly all of the structure has been confirmed by two to six independent sequence determinations on both DNA strands. The length of the 16S rRNA chain inferred from the DNA sequence is 1541 nucleotides, in close agreement with previous estimates. We note discrepancies between this sequence and the most recent version of it reported from direct RNA sequencing [Ehresmann, C., Stiegler, P., Carbon, P. & Ebel, J.P. (1977) FEBS Lett. 84, 337-341]. A few of these may be explained by heterogeneity among 16S rRNA sequences from different cistrons. No nucleotide sequences were found in the 16S rRNA gene that cannot be reconciled with RNase digestion products of mature 16S rRNA.

Base Sequence

Processing of the 5' end of Escherichia coli 16S ribosomal RNA.

We have isolated and partially characterized an endonuclease involved in processing the 5' end of 16S rRNA of Escherichia coli. A mutant strain that is deficient in this enzyme accumulates a new precursor of 16S rRNA, named 16.3S rRNA. This rRNA has the 3' end of mature 16S rRNA but is about 60 nucleotides longer at the 5' end. In vitro, the enzyme preparation cleaves an RNA fragment of about 60 nucleotides from the 5' end of 16.3S rRNA in 30S ribosomal subunits, yielding the mature 5' end of 16S rRNA. In the mutant strain the 16.3S rRNA is associated with a full complement of 21 ribosomal proteins in 30S subunits. These particles, which comprise 50% of the total 30S subunits, are present on polyribosomes.

Escherichia coli

Specific in situ cleavage of 16S ribosomal RNA of Escherichia coli interferes with the function of initiation factor IF-1.

Specific in situ cleavage of 16S rRNA of E. coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13. The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered. This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1. The initiation complexes formed with defective ribosomes are fully reactive with puromycin. Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied. Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.

Bacteriocins

Conformational changes in 16S ribosomal RNA induced by 30S ribosomal subunit proteins from Escherichia coli.

Laser light scattering has been used to evaluate conformational differences between free 16S RNA and several specific protein-16S RNA complexes. Proteins that interact strongly with the 16S RNA early in subunit assembly stabilize the RNA chain against unfolding in 1 mM Mg2+ and actually promote the formation of a more compact teriary structure in 20 mM Mg2+. A vital function of these proteins may therfore consist in altering the configuration of the RNA so that further assembly reactions can take place.

Bacterial Proteins

Reverse transcriptase pauses at N2-methylguanine during in vitro transcription of Escherichia coli 16S ribosomal RNA.

A restriction fragment strand complementary to a sequence near the 3' end of Escherichia coli 16S rRNA has been used to prime reverse transcriptase (avian myeloblastosis virus RNA-directed DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). In addition to transcripts that were extended to the 5' end of the RNA, two major transcription intermediates were observed. These discrete-sized cDNA intermediates are the result of a kinetic barrier imposed by monomethylation of the amino group on guanine that participates in base-pairing. Both major transcription intermediates correspond to attenuation at the known positions of N2-methylguanine (m2G) in the rRNA sequence. The relaxation time for elongation of the cDNA through m2G is approximately 3 min. No other major kinetic pauses were observed in the 1340 bases transcribed.

Base Sequence

The 3' terminal oligonucleotide of E. coli 16S ribosomal RNA: the sequence in both wild-type and RNase iii- cells is complementary to the polypurine tracts common to mRNA initiator regions.

Application of Sanger techniques to the analysis of the 3' terminal oligonucleotide from E. coli 32-P-labelled 16 S rRNA yields the sequence AUCACCUCCUUAOH. This sequence is identical in RNA isolated from two wild-type strains (MRE600 and E. coli B, SY106) and from a mutant strain (AB301/105) defective in RNase III. Data presented here explains the previous derivation of an incorrect sequence (AUCCUCACUUCAOH) by others. The functional significance of complementarity between the 3' terminus of 16S rRNA and poly-purine tracts commonly found in mRNA initiator regions is discussed.

Base Sequence

Synthesis of oligonucleotides with sequences identical with or analogous to the 3'-end of 16S ribosomal RNA of Escherichia coli: preparation of m-6-2-A-C-C-U-C-C and A-C-C-U-C-m-4-2C via phosphotriester intermediates.

The synthesis of two fully-protected hexanucleotides (11a and 11b) via a phosphotriester approach, which is based on the use of two types of protecting groups for the internucleotide linkages, i.e. one 2,2,2-tribromo-ethyl at the 5'-terminus and four 2-chlorophenyl groups for the remaining linkages, is reported. The hexanucleotides 11a and 11b, assembled via a block-wise two-step phosphotriester method, can be deblocked conveniently to give the two hexamers 12a and 12b containing only 3'leads to5' internucleotide linkages.

Adenine Nucleotides

Synthesis of oligonucleotides with sequences identical with or analogous to the 3'-end of 16S ribosomal RNA of Escherichia coli: preparation of A-C-C-U-C-C via the modified phosphotriester method.

A combination of two different methods for the synthesis of oligoribonucleotides, i.e. the two-step phosphotriester method with 2-chlorophenyl phosphate as bifunctional phosphate source and the modified triester method with 2,2,2-trichloroethyl 2-chlorophenyl phosphorochloridate as monofunctional phosphate source, is applied for the synthesis of the fully-protected hexaribonucleotide A-C-C-U-C-C. The two-step method is used for the synthesis of the required dinucleotide monophosphates 9, 10 and 11. Application of the modified triester method for the coupling of the oligonucleotide blocks results in the formation of the fully-protected hexamer 15. Furthermore, attention is paid to 2,4,6-triisopropylbenzenesulphonyl 4-nitroimidazolide as a new condensing agent for the coupling of larger oligonucleotide blocks.

Adenine Nucleotides

Phylogenetic origin of the chloroplast and prokaryotic nature of its ribosomal RNA.

The 16S ribosomal RNA of the Euglena gracilis chloroplast has been characterized in terms of its two-dimensional electrophoretic "fingerprint" (T1 ribonuclease). Results show it to be a typically prokaryotic 16 S rRNA. By the present criterion, different chloroplasts are shown to be related to one another and at least distantly to blue-green algae and perhaps to Bacillaceae. These results argue in favor of an endosymbiont origin of the chloroplast.

Base Sequence