Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene (21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and base-pair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.     

Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to alveolates

The pellicles of alveolates (ciliates, apicomplexans, and dinoflagellates) share a common organization, yet perform very divergent functions, including motility, host cell invasion, and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1,173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as green fluorescent protein-fusion constructs confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I, and V). When the T. thermophila novel proteins were compared with apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle and in doing so identified two new proteins of the apicomplexan invasive structure--the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these proteins might underpin the diversity and utility of their unique pellicular structure.     

Characterization of the T, L, I, S, M and P cell surface (immobilization) antigens of Tetrahymena thermophila: molecular weights, isoforms, and cross-reactivity of antisera.

In the ciliate protist Tetrahymena thermophila the L, H, T, I, S, M and P cell surface proteins (immobilization antigens) are expressed under different conditions of temperature (L, H, T), culture media (I, S), and mutant genotype (M, P). Immunoblot and autoradiographic studies using antisera to purified protein show that the molecular weights of these proteins range from 25,000 to 59,000. The H, T, S, M and P antigens are recognized as single polypeptides, whereas L, I, and one allelic form of T each appear to consist of a family of polypeptides. Although antisera are specific in immobilization and immunofluorescence assays of surface protein in living cells, cross-reactivity is seen with denatured protein on immunoblots. It is hypothesized that the surface protein genes are organized into families of evolutionarily related isoloci.     

Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins

Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins. The antisera were raised against either groups of r-proteins or individually purified r-proteins. Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D. melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D. melanogaster r-proteins (anti-TP80). The specificity of the antisera and the identity of the homologous E. coli r-proteins were characterized by using immunooverlay and immunoblot assays. These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D. melanogaster ribosomal proteins. The E. coli protein homologous to D. melanogaster protein S14 was identified as E. coli protein S6. By adsorption of the anti-TP80 serum, we determined that D. melanogaster protein 7/8 is homologous to the acidic E. coli protein L7/L12. D. melanogaster acidic protein 13 was also shown to be immunologically related to D. melanogaster protein 7/8.This research was supported by National Institutes of Health Grant GM23410 awarded to WYC. LMS was the recipient of a predoctoral fellowship from Molecular, Cellular, and Developmental Biology Training Grant PHS T32 CM07227. We are very grateful to Dr. Anthony Mahowald for providing us with embryos.     

In vivo stability, maturation and relative differential synthesis rates of individual ribosomal proteins in Escherichia coli B/r

The relative differential synthesis rates² of individual ribosomal proteins (r-proteins) were determined for Escherichia coli B/r growing in succinate medium (growth rate, μ = 0.65 doublings per hour), glucose medium (μ = 1.36) and glucose-amino acids medium (μ = 1.90). These differential synthesis rates were found to increase co-ordinately with increasing bacterial growth rates; this implies that ribosomes from bacteria growing at different rates are homogeneous with respect to their protein composition (i.e. the stoichiometric amounts of the different r-proteins per ribosome are constant and independent of the bacterial growth rate). Following incorporation into ribosomes, the bulk of the r-proteins were found to be as stable as total protein. Only two r-proteins, S6 and S21, were less stable than total protein; their decay half-lives, measured in succinate and glucose-amino acids cultures, were estimated to be approximately 500 minutes. In addition, post-translational modification of proteins S18, L6 and L11 was observed and the possible relations between modification and in vivo ribosome assembly are discussed. Finally, evidence is presented suggesting that the coordinate production of r-proteins may result, in part, from a mechanism that degrades excess r-proteins that are not rapidly incorporated into ribosomal particles.