GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.

The GTPase superfamily: conserved structure and molecular mechanism

An auxotrophic mutant which requires a metabolite not available in vertebrate tissues should be unable to grow in such tissue and thus be non-virulent. Most mutations to auxotrophy do not affect virulence1–3, presumably because the required metabolites are available at sufficient concentration. However, Bacon et al.1,2 noted that a Salmonella typhi mutant requiring p-amino-benzoic acid (p AB) was less virulent (for mice) than its parent. Salmonella, unlike vertebrates, cannot assimilate exogenous folate and must synthesize it from p AB; the virtual absence of p AB from vertebrate tissues is shown by the efficacy of sulphonamide chemotherapy. Yancey et al.4 reported reduced virulence for a S. typhimurium mutant with a requirement for 2,3-dihydroxybenzoate (DHB), the precursor of the bacterial iron-acquisition compound, enterochelin. As DHB is not a known vertebrate metabolite, it would be expected to be absent from mouse tissues. Salmonella synthesize p AB and DHB from chorismate, the final product of the aromatic biosynthetic (aro) pathway. A complete block at any step of this pathway should make S. typhimurium auxotrophic for two compounds not available in vertebrate tissues, and thus non-virulent. We describe here the use of a tetracycline-resistance transposon, Tn10 (refs 5, 6), inserted in gene aroA to produce non-reverting, aromatic-requiring derivatives of virulent S. typhimurium strains. These derivatives were virtually non-virulent; their use as live vaccines conferred excellent protection against challenge with a virulent strain.

Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines.

The complete general secretory pathway in gram-negative bacteria

http://pu.edu.pk/codon/articles/Codon%20Bias%20and%20Heterologous%20Protein%20Expression.pdf

Codon bias and heterologous protein expression

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

/pdf/comparative-genomics-of-the-lactic-acid-bacteria-43g3cc7xfw.pdf

Comparative genomics of the lactic acid bacteria

Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.

/pdf/improvement-of-reading-frame-maintenance-is-a-common-4y1jrc8e99.pdf

Improvement of reading frame maintenance is a common function for several tRNA modifications.

Transfer RNA (tRNA) from all organisms on this planet contains modified nucleosides, which are derivatives of the four major nucleosides. tRNA from Escherichia coli/Salmonella enterica contains 31 different modified nucleosides, which are all, except for one (Queuosine[Q]), synthesized on an oligonucleotide precursor, which through specific enzymes later matures into tRNA. The corresponding structural genes for these enzymes are found in mono- and polycistronic operons, the latter of which have a complex transcription and translation pattern. The syntheses of some of them (e.g.,several methylated derivatives) are catalyzed by one enzyme, which is position and base specific, but synthesis of some have a very complex biosynthetic pathway involving several enzymes (e.g., 2-thiouridines, N6-threonyladenosine [t6A],and Q). Several of the modified nucleosides are essential for viability (e.g.,lysidin, t6A, 1-methylguanosine), whereas deficiency in others induces severe growth defects. However, some have no or only a small effect on growth at laboratory conditions. Modified nucleosides that are present in the anticodon loop or stem have a fundamental influence on the efficiency of charging the tRNA, reading cognate codons, and preventing missense and frameshift errors. Those, which are present in the body of the tRNA, have a primarily stabilizing effect on the tRNA. Thus, the ubiquitouspresence of these modified nucleosides plays a pivotal role in the function of the tRNA by their influence on the stability and activity of the tRNA.

Transfer RNA Modification

Modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U), were found in nucleic acids as early as 1948. Modified nucleosides are contained in tRNA from all three phylogenetic domains—Archaea, Bacteria, and Eucarya—which were formerly called the kingdoms of archaebacteria, eubacteria, and eukaryotes, respectively. Although modified nucleosides are found in various positions in the tRNA, two positions, 34 and 37, contain the largest variety of modified nucleosides. This chapter presents an overview of the coding properties associated with modified nucleosides present in positions 34 and 37. Outside the anticodon, the modified nucleosides are usually “simple” modifications like methylated or thiolated derivatives, whereas all except one (archaeosine in tRNAs from Archaea) of the hypermodified nucleosides are present in the anticodon region and only in positions 34 and 37. Moreover, outside the anticodon region, only one or two kinds of modified nucleosides in each position are present, whereas a large variety of modified nucleosides are present in the anticodon region, especially in positions 34 and 37. During evolution, structural refinements of the individual tRNAs were presumably fulfilled by the evolution of the synthesis of the various modified nucleosides, including the hypermodified nucleoside. Modified nucleosides in the anticodon region exert their functions primarily in the decoding process, whereas modified nucleosides outside this region may primarily be involved in other tRNA interactions, such as interactions with translation factors or as sensors for environmental stress conditions.

Biosynthesis and Function of Modified Nucleosides

The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic c ...

Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation. The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Δ cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Δ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

/pdf/the-essential-gcd10p-gcd14p-nuclear-complex-is-required-for-4acpdnbq9u.pdf

Glenn R. Björk

Papers

Improvement of reading frame maintenance is a common function for several tRNA modifications.

Transfer RNA Modification

Biosynthesis and Function of Modified Nucleosides

Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.

The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA