End Of 16S rRNA Research Articles

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A 2660, which lies in a sequence that is highly conserved in all species.

Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli

Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes. The interaction of these labeled RNAs with the 30S subunit of Escherichia coli was studied by using fluorescence spectroscopic techniques. All the RNAs bound tightly to 30S subunits (Kd less than or equal to 200 nM). Resonance energy transfer experiments demonstrated the proximity of the ends of the RNAs to each other and to two fluorescently labeled sites on the 30S subunit: the 3' end of 16S rRNA and the cysteine residue of ribosomal protein S21. By using the distances calculated from energy transfer between the 3' end of 16S rRNA and the ends of RNAs of varying lengths, a topological map of this region of mRNA on the 30S subunit was constructed.

Influence of the second and third codon on the expression of recombinant hirudin in E. coli

The effect of all possible codons corresponding to the second and third amino acid (isoleucine and threonine) on the expression level of hirudin in E. coli has been analysed. These levels could not be correlated with changes in primary and secondary mRNA structure. A decrease in the rate of synthesis and of product accumulation follows the introduction for ile of the ATA codon which is of very low usage, and for thr of the ACC codon, which results in homology of the mRNA with the 3'-end of 16S rRNA. The results are discussed according to current concepts of protein expression in E. coli.

Translation by the adenovirus tripartite leader: elements which determine independence from cap-binding protein complex

The adenovirus tripartite leader is a 200-nucleotide-long 5' noncoding region which facilitates translation of viral mRNAs at late times after infection. The tripartite leader also confers the ability to initiate translation independent of the requirement for cap-binding protein complex or eIF-4F without any requirement for adenovirus gene products. To elucidate the manner by which the tripartite leader functions, the primary determinants of leader activity were investigated in vivo by testing a series of mutations expressed from transfected plasmids. The results of these experiments indicate that the tripartite leader does not promote internal ribosome binding, at least in a manner recently described for picornavirus mRNAs. In addition, despite an unusual arrangement of sequences complementary to the 3' end of 18S rRNA in the tripartite leader, we could find no evidence for involvement in its translation activity. Instead, our results are consistent with a model in which much of the first leader is maintained in an unstructured conformation which determines the ability of the tripartite leader to facilitate translation and bypass a normal requirement for eIF-4F activity. Several possible translation models are discussed, as well as the implications for translation of late viral mRNAs.

Structural analysis of the short length ribosomal DNA variant fromPisum sativumL cv. Alaska

The genomic clone, RRNpss1, representing the short ribosomal DNA length variant in Pisum sativum L. cv. Alaska, has been isolated and the 2859 bp intergenic spacer, along with the 25S rRNA 3' border and 18S rRNA 5' border, has been sequenced. The intergenic spacer contains nine tandem repeats, approximately 180 bp in length, which show greater than 80% sequence homology to each other. The RNA polymerase I transcription start site and a processing site, located 776 bp and 536 bp upstream of the 5' end of 18S rRNA, respectively, have been determined using S1 analysis. The region surrounding the +1 site shows strong homology between the positions -6 to +10 to the rDNA sites of initiation in radish, maize, and wheat. The sequence CATGCAAA is located 19 bp upstream of the site of initiation, and appears once within each subrepeat and twice more between the end of the subrepeat array and the site of initiation. A previously characterized HpaII site which shows developmental regulation of methylation is located 31 bp downstream of the site of initiation. Using RFLP linkage analysis, the short rDNA length variant of cv. Alaska is assigned to Chromosome 4 where it is genetically independent of the long rDNA length variant which is putatively assigned to Chromosome 7.

Accurate processing of human pre-rRNA in vitro.

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

Accurate processing of human pre-rRNA in vitro.

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

Determination and evolutionary significance of nucleotide sequences near to the 3′-end of 16S ribosomal RNA of mycobacteria

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

A nuclease that cuts specifically in the ribosome binding site of some T4 mRNAs.

We have identified a nucleolytic activity in Escherichia coli infected by bacteriophage T4 that introduces cuts in the ribosome binding site of at least two T4 mRNAs. Cutting takes place specifically in the GGAG sequences that are complementary to the 3' end of 16S rRNA (Shine-Dalgarno sequence). The nature of this nucleolytic cut has been investigated by reverse transcriptase mapping, anti-mRNA mapping, utilization of the vaccinia virus guanylyltransferase, and labeling by polynucleotide kinase. We have compared the sequences of target mRNAs with an mRNA of similar sequence but that is not a substrate for the nuclease. This allowed us to narrow down the possibilities for sequence elements that determine nuclease recognition. We hypothesize that this nuclease plays a physiological role in the inhibition of expression of a class of phage proteins.

Escherichia coli 3′-terminal 16S rRNA sequence modulated fidelity during translation

The ribosome is a central component of the protein synthetic apparatus. Although progress has been made in characterizing the functional role of many of the ribosomal proteins, the properties of ribosomal RNA and its role in ribosome structure and function are not well understood. To investigate the working properties of the highly conserved 3′-end of 16S rRNA, a site-specific deletion was made directly within the 16S rRNA molecule. The terminal deletion did not impair in vitro 30S subunit assembly, but the particles produced lost translational fidelity in an in vitro translation system primed with natural mRNA.

Selection of the mRNA translation initiation region by Escherichia coli ribosomes.

Two genes specifying model mRNAs of minimal size and coding capacity, with or without the Shine-Dalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka approximately 2 x 10(7) M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the initiator tRNA.

Reading frame switch caused by base-pair formation between the 3′ end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli.

Watson-Crick base pairing is shown to occur between the mRNA and nucleotides near the 3' end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base-pairing is similar to the mRNA-rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S-D) nucleotides of ribosome binding sites and their complements in the 1540-1535 region of 16S rRNA. mRNA-rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S-D nucleotides precisely spaced 5' on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S-D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3' end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.

A temperature-sensitive mutant in the gene rplX for ribosomal protein L24 and its suppression by spontaneous mutations in a 23S rRNA gene of Escherichia coli.

A temperature-sensitive mutant with an altered ribosomal protein L24 was analysed. Revertant analysis showed that the temperature-sensitive growth was correlated with the altered protein. A DNA segment containing the mutant rplX gene was cloned and sequenced. The GGC codon for glycine at the amino acid position 84 of the protein was found to be altered to a GAC codon for aspartic acid. By transforming the rplX mutant with a plasmid carrying the rrnB operon and by selecting for temperature-resistant transformants we obtained two spontaneous suppressor mutants in the gene for 23S rRNA. DNA sequence analysis of the region corresponding to the 5' end of the 23S rRNA showed a C to T alteration at position 33 in both mutants and an additional A to G alteration at position 466 in one of them. The results suggest intimate interaction of protein L24 and the 5' end of 23S rRNA in vivo and support a secondary structure model of the 23S rRNA which brings these mutational points into a close contact.

Circular dichroism and 500-MHz proton magnetic resonance studies of the interaction of Escherichia coli translational initiation factor 3 protein with the 16S ribosomal RNA 3' cloacin fragment.

The RNA helix destabilizing properties of Escherichia coli initiation factor 3 protein (IF3), and its affinity for an evolutionarily conserved sequence at the 3' end of 16S rRNA, led us to examine the details of the protein-nucleic acid interactions upon IF3 binding to the 49-nucleotide 3'-terminal cloacin DF13 fragment of 16S rRNA by studying the circular dichroism (CD) and proton magnetic resonance spectra of the RNA, the protein, and their complex. In a physiological tris(hydroxymethyl)aminomethane buffer, where the interaction is primarily nonionic and sequence specific, addition of IF3 decreases the RNA 268-nm CD peak hyperbolically by 19% to an end point of about one IF3 per RNA strand. The titration curve is best fit by an association constant of (1.80 +/- 0.05) X 10(7) M-1, within the range estimated by a nuclease mapping study of the same system [Wickstrom, E. (1983) Nucleic Acids Res. 11, 2035-2052]. In a low-salt phosphate buffer without Mg2+, where the interaction is primarily ionic and nonspecific, titration with IF3 decreases the peak CD sigmoidally by 35% to an end point of two IF3 per strand. The titration curve is best fit by an intrinsic association constant of (1.7 +/- 0.7) X 10(6) M-1 for each IF3 and a cooperativity constant of 33 +/- 6. In a physiological phosphate buffer lacking Mg2+, the dispersion of aromatic proton magnetic resonance peaks and upfield-shifted methyl proton resonances indicates a high degree of secondary and tertiary structure in the protein. In an equimolar mixture of IF3 and RNA cloacin fragment, several changes in identifiable IF3 and RNA resonances are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Part of the human ribosomal RNA locus stabilizes a plasmid in yeast

Most yeast plasmids--particularly those containing chromosomal replicators (ARS)--are unstable and do not segregate equally to mother and daughter cells unless they contain centromeric sequences. We have screened a fraction of the human genome for sequences that stabilize YRp7, a plasmid containing ARS1. We selected a fraction which we hoped would be enriched in human centromeric sequences--the DNA attached to the nucleoskeleton. We obtained one human sequence that partially stabilized a yeast plasmid and, surprisingly, it contained sequences homologous to those coding for the 3' end of 18s rRNA, the transcribed spacer and 5' end of 28s rRNA. This sequence did not show any ARS activity nor did it increase the copy number of the plasmid and so probably improved partition of the plasmid between mother and daughter cells. It had no homology to yeast centromeres.

Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus.

The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA.

Non-repetitive AT-rich sequences are found in intergenic regions of Methanococcus voltae DNA.

AT-rich DNA, which constitutes a distinct fraction of the cellular DNA of the archaebacterium Methanococcus voltae, was shown to consist of non-repetitive sequences dispersed on the chromosome and to lack continuous open reading frames in five out of six randomly analyzed cases. Upon subsequent analysis of intergenic regions, AT-rich sequences were again detected. Transcription start points were mapped in front of three open reading frames. The 5' ends of the transcripts were found in variable positions relative to the AT-rich sequences in the different cases. Shine-Dalgarno type sequences complementary to the 3' end of 16S rRNA were discovered at suitable distances from the 5' ends of the genes.

Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences.

Basepairing of oligonucleotides to the 3' end of 16S ribosomal RNA is not stabilized by ribosomal proteins.

We have studied the binding of the octanucleotide (5'-3')d(AAGGAGGT) which is fully complementary to the 3' end of 16S ribosomal RNA, to ribosomes and to the isolated target sequence (5'-3') (ACCUCCUUA). The binding constant for 30S or 70S ribosomes is (5 +/- 2) X 10(7) mol-1, whereas the duplex containing the octa- and the nonanucleotide has an association constant of (6 +/- 3) X 10(7) mol-1. The two values are the same within the experimental error. This result suggests that basepairing at the 3' end of 16S rRNA is not stabilized by ribosomal proteins.