Chapter 3 : Structure and Expression of Prokaryotic tRNA Genes

Screen for E. coli tRNA genes: example of autoradiogram from screen for clones encoding tRNAs. Four gels (20 samples each) analyzing clones 101 to 180 from Kohara's miniset of λ clones ( 22 ) are shown. 32P-labeled RNA samples were prepared as follows: cultures of E. coli strain CA274, heavily irradiated with UV light (3 min at a dose of 10 ergs/mm2 per second), were infected with each phage clone (multiplicity of infection, about 10) and labeled with [32P]orthophosphate in a scaled-down version (25 ml per sample) of the method previously described ( 19 ). After a 3-h incubation at 37°C in the presence of chloramphenicol (150μg/ml), 32P-labeled RNAs were isolated from the infected cultures by phenol extraction and were separated by electrophoresis in 10% (wt/vol) polyacrylamide gel slabs (45 cm long, 30 cm wide, and 0.35 mm thick) containing 50 mM Tris-borate buffer (pH 8.3) and 8 M urea. The serial numbers of clones that encode tRNAs are shown. Arrows indicate 5S rRNA and tRNA. The asterisk indicates a partial transcript of the λ N gene. This band indicated a successful infection by each clone and acted as an internal size marker, dividing the bands of tRNAs from 5S rRNA and other small RNAs on the autoradiogram. The top scale indicates physical map locations.

Comprehensive map of the tRNA genes in E. coli K-12. The tRNA gene designations specify the various isoacceptor species encoded and their order of transcription. Designations for tRNA genes in the rRNA operons are followed by the name of the operon in parentheses, in which each operon is indicated. Small arrows inside the outer circle indicate the orientation of each transcriptional unit. The numbers in the outer circle indicate the scale of the physical map in kilobases. The numbers in the inner circle are the corresponding scale of the genetic map in minutes (min). The replication origin (oriC) and termination (terC) sites are indicated. Although the information in this map was based on studies using Kohara's clone library, a large inversion was found in Kohara's physical map for strain W3110; the correct orientation of the chromosome is depicted here.

Numbers of isoacceptors in M. capricolum. M. capricolum tRNAs specific for individual amino acids were labeled by Andachi et al. ( 1 ) according to the method of Traboni et al. ( 50 ). Deacylated total tRNAs (100 μg) of M. capricolum were aminoacylated at 30°C for 1 hour, with each amino acid present at 120 mM in a reaction mixture (300 μl) containing 100 mM sodium cacodylate (pH 7.0), 10 mM magnesium acetate, 10 mM KC1, 2 mM ATP, 2 mM β-mercaptoethanoI, and 30 μl of M. capricolum S-100 fraction. The tRNAs were recovered from the mixture by extraction with phenol and incubated in 50 μl of 100 mM sodium metaperiodate (pH 4.5) for 30 min at room temperature in the dark to oxidize ribose at the 3′ end of nonaminoacylated tRNAs, leaving aminoacylated tRNA unoxidized. The reaction was stopped by the addition of 5 μl of 1 M rhamnose, and incubation was continued for 30 min at room temperature in the dark. After precipitation with ethanol, the tRNAs were incubated in 100 μl of 0.5 M lysine (pH 8.8) at 37°C for 30 min for β-elimination of oxidized, or for deacylation of unoxidized, tRNAs. For selective 3′ end labeling of unoxidized tRNA species, total tRNAs (2 μg) treated as described above were incubated at 4°C for 2 hours with 10 μCi of 5′-[32P]pCp (3,000 Ci/mmol) and 1 unit of phage T4 RNA ligase in a volume of 10 μl, as described by England et al. ( 10 ). The labeled tRNA for each of the 20 amino acids was separated by electrophoresis with 12% polyacrylamide-7 M urea gel and exposed on an x-ray film.

Organization of tRNA gene clusters. Tandemly arrayed tRNA genes in E. coli (a) and M. capricolum (b). Individual tRNA genes are represented by hatched boxes. The symbols P, AT, and T indicate promoter, attenuator, and terminator of transcription, respectively. The anticodons of each tRNA species are shown in parentheses as DNA sequences. The numbers represent base pairs between the tRNA genes. The position of each cluster on the linkage map of E. coli ( 3 ) is shown in the hollow box preceding each array.

Structures of rRNA operons of E. coli K-12. The overall organization of the seven rrn operons is represented schematically in the upper part of the figure. The brackets mark tRNA or 5S RNA genes that are not common to all operons. The direction of transcription is from left to right. The identities of the known tRNA genes are given for each operon in the lower portion of the figure. Sequenced tRNA genes are identified with bold letters.

DNA sequence similarity of 16S and 23S rRNA genes in rrnH and rrnD operons. Stars indicate the bases identical in both sequences. Stippled regions represent coding regions for the 16S and 23S rRNA genes (partial) and the spacer tRNAs (tRNA1 Ile and tRNA1B Ala).

Mixed-function operons containing tRNA and protein-encoding genes in E. coli. The open boxes represent the open reading frame (ORF) or gene indicated. For explanation of symbols, see Fig. 4 legend.

Comparison of tRNA gene promoters and prokaryotic promoter consensus sequence. The overall organization and sequences of the promoter elements are shown in schematic form in the upper part of the figure. Distances between the elements and tRNA gene (stippled box) are not drawn to scale. The numbers in the lower portion of the figure indicate the numbers of tRNA gene promoters carrying the consensus residue at each position; 34 E. coli promoters and 14 M. capricolum promoters were compared.

Repeat structures found in 3′ regions of tRNA genes and in distal flanking sequences. Arrows indicate repeated sequences. Dashed arrows indicate weak homology. The stippled part of the tRNA gene represents the 18 to 19 bp of the 3′ terminal region of tRNA. The minutes (min) correspond to those of the genetic map of E. coli ( Fig. 2 ).

Temperate phage attachment sites in the 3′ regions of tRNA genes. Integration occurs by recombination in the hatched region. The bold line represents the phage genome. The putative products of several integration events are represented in the lower portion of the figure.

Correlation between tRNA levels and amino acid usage. Relative tRNA content for each amino acid in E. coli (closed circle) and M. capricolum (open circle) is plotted against the usage of each amino acid in proteins. (Values taken from Table 2 .)