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Publications and source records attributed to V A Erdmann.
Crystals of purified 5 S rRNA from Thermus flavus have been obtained. The crystals diffract up to 8 A resolution, using synchrotron radiation, and have the monoclinic space-group C2. The unit cell has the dimensions a = 190 A, b = 110 A, c = 138 A and beta = 117 degrees. The cell volume suggests the presence of four 5 S rRNA molecules per asymmetric unit.
The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.
This is an update for the 5S rRNA sequences of the BERLIN RNA DATABANK last published in 1990 (1). The new entry consists of 25 eubacterial and 2 eukaryotic 5S rRNA sequences and 10 plant 5S rRNA pseudogenes (Table 1). Thus the BERLIN RNA DATABANK contains as of February 1, 1991 the 5S rRNA sequences of 44 archaebacteria, 292 eubacteria, 20 plastids, 6 mitochondria, 321 eukaryotes and 21 eukaryotic pseudogenes. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information.
The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.
The two 23S rRNA-5S rRNA-tRNAGly operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterized the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGly primary transcripts in this organism by nuclease S1 mapping. A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A. Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events. Termination of transcription at the uridine cluster following the termination-associated hairpin was shown to be efficient but leaky. Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region. The promoter directed synthesis of a beta-galactosidase fusion protein in Escherichia coli.
A model for the tertiary structure of plant 5S rRNA, previously proposed by our laboratory (Joachimiak, A. et al. (1990) Int. J. Biol. Macromol., in press) was tested by specific cleavage of the plant 5S rRNA in the presence of synthetic oligodeoxynucleotides. The hexanucleotides used in this study were complementary to different portions of loops C, D and E, the nucleotides of which have recently been proposed to be involved in tertiary hydrogen bonds. The results obtained strongly support the interaction of loops C and D by nucleotides C34, C35, C36, A37 and G85, G86, G87, U88, respectively. Digestion pattern of loop E (domain gamma, nucleotides 66-110) suggests a possible different arrangement of this part of the plant 5S rRNA molecule, when compared with other eukaryotes.
Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant, stable RNA identified in the Gram-positive eubacterium Bacillus subtilis. Several findings suggest an important role of scRNA in protein biosynthesis: it shares structural and biochemical features with the Escherichia coli 4.5S RNA (114 nucleotides), a molecule known to be involved in this process, and it can complement the essential function of 4.5S RNA in vivo. The common apical hairpin motif of scRNA and 4.5S RNA also exists in eukaryotic 7SL RNA, the RNA component of the signal recognition particle. To elucidate the higher-order structure of scRNA, we have combined a phylogenetic approach with a biochemical one. The sequence of scRNA from a thermophilic relative of B. subtilis, Bacillus stearothermophilus, was determined and compared with the B. subtilis scRNA. In addition, the solution structure of B. stearothermophilus scRNA was probed with single- and double-strand-specific nucleases. Both types of analysis support a secondary structure model for scRNA that strongly resembles 4.5S RNA and respective parts of 7SL RNA. The results provide further evidence for the suggestion of a functional relationship between these RNAs.
The coding region for the cytotoxin alpha-sarcin from Aspergillus giganteus has been chemically synthesized by the ligation of 19 overlapping oligodeoxyribonucleotides. An Escherichia coli clone producing the cytotoxin was constructed by inserting the synthesized gene directly downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-OmpA2. The enzyme encoded by the chemically synthesized gene expressed in E. coli displayed properties identical to those of native alpha-sarcin isolated from A. giganteus with respect to its chemistry, antigenicity and ribonucleolytic activity in qualitative assays.
The Bacillus subtilis small cytoplasmic RNA (scRNA) is the structural homologue of both the RNA component of the eukaryotic signal recognition particle (SRP) and the Escherichia coli 4.5S RNA, and it can complement the essential function of the latter RNA in vivo. In the course of characterization of the single-copy scRNA gene locus (scr) we identified an open reading frame, termed ORF17, upstream from scr that encodes an acidic 17 kDa protein of unknown function. This analysis involved DNA sequencing, monitoring expression of transcriptional and translational ORF17-cat and ORF17-lacZ fusions, respectively, and purification and sequencing of the ORF17-lacZ fusion protein. Apparently, transcription of ORF17 proceeds into scr. A small portion of the 17 kDa protein shows homology to deoxycytidylate (DCMP) deaminase of bacteriophagphage T2, but no similarity exists to the sequenced SRP-polypeptides or any other known protein sequences.
The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.
The cytotoxin alpha-sarcin was employed to test the model of secondary and tertiary structures of plant 5S rRNAs, which we recently proposed [(1990) Int. J. Biol. Macromol. (in press)]. alpha-Sarcin is a novel ribonuclease that hydrolyzes phosphodiester bonds adjacent to purines in nucleic acids. The digestion pattern obtained for lupin and wheat germ 5S rRNAs strongly suggests the existence of tertiary interactions between residues C34, C35, C36, A37 and G85, G86, G87, U88 as previously proposed. The results on the secondary structure of plant 5S rRNA are in line with a previously proposed model.
The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for beta and beta', 40 kDa for alpha and 92 kDa for sigma. The RNA polymerase is active at elevated temperatures (65 degrees C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping.
The BERLIN RNA DATABANK as of December 31, 1989, contains a total of 667 sequences of 5S rRNAs or their genes, which is an increase of 114 new sequence entries over the last compilation (1). It covers sequences from 44 archaebacteria, 267 eubacteria, 20 plastids, 6 mitochondria, 319 eukaryotes and 11 eukaryotic pseudogenes. The hardcopy shows only the list of those organisms whose sequences have been determined. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information.
The Bacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog to dnaZX of Escherichia coli, termed 'dnaX'. The scRNA gene region of a B. subtilis wild-type strain was marked with a cat gene and mapped by scoring chromosomal cotransformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with an EcoRI map comparison, places the scRNA gene and dnaX in the vicinity of recM near the replication origin region of B. subtilis.
The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.
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The essential 4.5S RNA of Escherichia coli contains a structural motif, which is also present in RNAs from other organisms, i.e. Bacillus subtilis scRNA, Halobacterium halobium 7S RNA and eukaryotic 7SL RNAs. This suggests a common function in all organisms, which could be related to protein translocation, since 7SL RNA is essential for this process in eukaryotes. We have analysed the structure and expression of the 4.5S RNA gene from another gram-negative eubacterium, Pseudomonas aeruginosa. The single copy gene encodes a 113 nucleotides long RNA, which shares 75% sequence homology to the E. coli 4.5S RNA and also exhibits the completely conserved hairpin structure of the corresponding RNAs of B. subtilis and E. coli. Transcription initiates 24 nucleotides upstream from the mature 5' end and exceeds beyond the 4.5S RNA coding region. A distal open reading frame, similar to that described for E. coli, does not exist downstream from the P. aeruginosa 4.5S RNA gene.
The 271 nucleotides long scRNA (small cytoplasmic RNA) from Bacillus subtilis is structurally related to the Escherichia coli 4.5 S RNA (114 nucleotides), an essential molecule supposed to be involved in protein biosynthesis, but it possesses an additional moiety completely missing in the E. coli 4.5 S RNA. Both RNAs share a conserved hairpin with the eukaryotic 7SL RNAs, which mediate protein translocation as part of the signal recognition particle (SRP). We have cloned and sequenced the entire scRNA gene region from B. subtilis and have studied transcription and processing of the scRNA in B. subtilis by nuclease S1 mapping. This analysis revealed the scRNA gene to constitute a monofunctional transcription unit, expressed from a single promoter to a rho-independent terminator, yielding a precursor which extends the mature scRNA by approximately 40 nucleotides at both ends. Processing of the scRNA apparently involves only two endonucleolytic cuts and occurs first at the 5' end.