The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast.

Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.

Heat shock induces a loss of rRNA-encoding DNA repeats in Brassica nigra.

Stress-induced mutations may play an important role in the evolution of plants. Plants do not sequester a germ line, and thus any stress-induced mutations could be passed on to future generations. We report a study of the effects of heat shock on genomic components of Brassica nigra Brassicaceae. Plants were submitted to heat stress, and the copy number of two nuclear-encoded single-copy genes, rRNA-encoding DNA (rDNA) and a chloroplast DNA gene, was determined and compared to a nonstressed control group. We determined whether genomic changes were inherited by examining copy number in the selfed progeny of control and heat-treated individuals. No effects of heat shock on copy number of the single-copy nuclear genes or on chloroplast DNA are found. However, heat shock did cause a statistically significant reduction in rDNA copies inherited by the F1 generation. In addition, we propose a DNA damage-reppair hypothesis to explain the reduction in rDNA caused by heat shock.

The "DEAD box" protein DbpA interacts specifically with the peptidyltransferase center in 23S rRNA.

The Escherichia coli DEAD (Asp-Glu-Ala-Asp) box protein DbpA is a putative RNA helicase and established RNA-dependent ATPase and is the only member of the DEAD box protein family for which a specific RNA substrate, bacterial 23S rRNA, has been identified. We have investigated the nature of this specificity in depth and have localized by deletion mutagenesis and PCR a single region of 93 bases (bases 2496-2588) in 23S rRNA that is both necessary and sufficient for complete activation of ATPase activity of DbpA. This target region forms part of the peptidyltransferase center and includes many bases involved in interaction with the 3' terminal adenosines of both A- and P-site tRNAs. Deletion of stem loops within the 93-base segment abolished ATPase activation. Similarly, point mutations that disrupt base pairing within stem structures ablated stimulation of ATPase activity. These data are consistent with roles for DbpA either in establishing and/or maintaining the correct three-dimensional structure of the peptidyltransferase center in 23S rRNA during ribosome assembly or in the peptidyltransferase reaction.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

The only essential function of TFIIIA in yeast is the transcription of 5S rRNA genes.

We have developed a system to transcribe the yeast 5S rRNA gene in the absence of the transcription factor TFIIIA. A long transcript was synthesized both in vitro and in vivo from a hybrid gene in which the tRNA-like promoter sequence of the RPR1 gene was fused to the yeast 5S RNA gene. No internal initiation directed by the endogenous 5S rDNA promoter or any processing of the hybrid transcript was observed in vitro. Yeast cells devoid of transcription factor TFIIIA, which, therefore, could not synthesize any 5S rRNA from the endogenous chromosomal copies of 5S rDNA, could survive if they carried the hybrid RPR1-5S construct on a multicopy plasmid. In this case, the only source of 5S rRNA was the precursor RPR1-5S transcript that gave rise to two RNA species slightly larger than wild-type 5S rRNA. This establishes that the only essential function of TFIIIA is to promote the synthesis of 5S rRNA. However, cells devoid of TFIIIA and surviving with these two RNAs grew more slowly at 30 degrees C compared with wild-type cells and were thermosensitive at 37 degrees C.

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.

Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes.

We have explored the evolutionary history of the Apicomplexa and two related protistan phyla, Dinozoa and Ciliophora, by comparing the nucleotide sequences of small subunit ribosomal RNA genes. We conclude that the Plasmodium lineage, to which the malarial parasites belong, diverged from other apicomplexan lineages (piroplasmids and coccidians) several hundred million years ago, perhaps even before the Cambrian. The Plasmodium radiation, which gave rise to several species parasitic to humans, occurred approximately 129 million years ago; Plasmodium parasitism of humans has independently arisen several times. The origin of apicomplexans (Plasmodium), dinoflagellates, and ciliates may be > 1 billion years old, perhaps older than the three multicellular kingdoms of animals, plants, and fungi. Digenetic parasitism independently evolved several times in the Apicomplexa.