Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris

A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes a polypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid-encoded genes. We have designated this protein NsRpoTp.     

Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.     

Effect of RyhB small RNA on global iron use in Escherichia coli

RyhB is a noncoding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single noncoding RNA on iron homeostasis.     

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.     

Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi

A catechcol-type siderophore, assigned the trivial name chrysobactin, was isolated from the phytopathogenic bacterium Erwinia chrysanthemi and characterized by degradation and spectroscopic techniques as N-[N2-(2,3-dihydroxybenzoyl)-D-lysyl]-L-serine. Chrysobactin, which was also obtained by chemical synthesis, was shown to be active in supplying iron to a group of mutants of E. chrysanthemi defective in biosynthesis of the siderophore.     

Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis

Aryl acid adenylation domains are the initial enzymes for aryl-capping of catecholic siderophores in a plethora of microorganisms. In order to overcome the problem of iron acquisition in host organisms, siderophore biosynthesis is decisive for virulence development in numerous important human and animal pathogens. Recently, it was shown that growth of Mycobacterium tuberculosis and Yersinia pestis can be inhibited in an iron-dependent manner using the arylic acyl adenylate analogue 5'-O-[N-(salicyl)-sulfamoyl] adenosine that acts on the salicylate activating domains, MbtA and YbtE [Ferreras JA, Ryu JS, Di Lello F, Tan DS, Quadri LEN (2005) Nat Chem Biol1, 29-32]. The present study explores the behaviour of the 2,3-dihydroxybenzoate activating domain DhbE (bacillibactin synthesis) and compares it to that of YbtE (yersiniabactin synthesis) upon enzymatic inhibition using a set of newly synthesized aryl sulfamoyl adenosine derivatives. The obtained results underline the highly specific mode of inhibition for both aryl acid activating domains in accordance with their natively accepted aryl moiety. These findings are discussed regarding the structure-function based aspect of aryl substrate binding to the DhbE and YbtE active sites.     

The regulation of protein synthesis by translation control RNA

The mechanism by which translational control RNA (tcRNA) inhibits protein synthesis was investigated. In the presence of heme the inhibitory role of muscle tcRNA on hemoglobin synthesis was confirmed. Upon the addition of muscle tcRNA to a rabbit reticulocyte cell-free system the binding of [³²P]-globin mRNA to 40S ribosomal subunits and its subsequent incorporation into polysomes was inhibited. Furthermore, muscle tcRNA inhibits met-tRNA binding to polysomes and yet stimulates the formation of methionine-puromycin. These results suggest that muscle tcRNA blocks the binding of globin mRNA to ribosomes resulting in an abortive initiation complex that is, however, still capable of the methionine-puromycin reaction.     

Yeast RNA helicases of the DEAD-box family involved in translation initiation

RNA helicases of the DEAD-box and related families have been found to be required for all processes involving RNA molecules. Biochemical and genetic analyses have shown that at least two RNA helicases are required for translation initiation in yeast. Although it is generally believed that these enzymes are necessary to unwind secondary structures in the 5' untranslated region of mRNAs, their exact role has not been convincingly shown. We discuss here our present knowledge of the function of eIF4A and Ded1p, two DEAD-box proteins required for translation in eukaryotic cells.     

Simultaneous synthesis,translation, and storage of mRNA including histone mRNA in sea urchin eggs

The kinetics of accumulation of RNA labeled with uridine and the time course of change in the specific activity of the UTP pool were used to estimate the rate constants for synthesis and decay of RNA synthesized in unfertilized eggs of the sea urchin Lytechinus pictus. The rate of synthesis per haploid genome is similar to that in embryos. Most of the RNA is turning over with a half-life of about 5 hr, and an average of 11 pg of newly synthesized RNA accumulates at steady state. About 3.7% of the RNA in the polysomes of the egg is newly synthesized and this RNA has the heterogeneous size distribution expected for mRNA. Thus most, probably all, of the mRNA translated in the egg is also synthesized in the egg. Little, if any, of the RNA synthesized in the egg enters polysomes following fertilization. Thus the egg synthesizes a population of mRNA which is unstable and translated, but it also contains a more stable, untranslated population of previously synthesized, stored mRNA, which is translated only after fertilization. Since the two populations of mRNA code for the same abundant proteins (Brandhorst, B. P. (1976). Develop. Biol., 52, 310–317), there is a temporal separation in the metabolism and function of coexisting mRNA molecules of identical coding sequence. Among the mRNAs synthesized and translated in the egg are histone mRNAs having the same electrophoretic mobilities and rates of synthesis per genome as those synthesized in rapidly cleaving embryos. Thus the synthesis, entry into the cytoplasm, and translation of histone mRNA are not restricted to the S phase of the cell cycle or the period of cell division.     

Remote control of mRNA cleavage by a small RNA

Comment on: Prévost K, et al. Genes Dev 2011; 25:385-96.