Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA 'suppressor'. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA(1)(fMet), and a distantly located metY gene encodes a variant, tRNA(2)(fMet). In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA(2)(fMet) with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA(+) host background. The mutations were observed when the plasmid-borne gene encoded tRNA(2)(fMet) (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/psi39G40A41). The usefulness of this strain, and a possible role for multiple tRNA(1)(fMet) genes in E. coli in safeguarding their intactness, are discussed.

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys48-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-α and CD3-δ.

Prokaryotes contain cytoskeletal proteins such as the tubulin-like FtsZ, which forms the Z ring at the cell center for cytokinesis [1], and the actin-like MreB, which forms a helix along the long axis of the cell and is required for shape maintenance [2]. Using time-lapse analysis of Escherichia coli cells expressing FtsZ-GFP, we found that FtsZ outside of the Z ring also localized in a helix-like pattern and moved very rapidly within this pattern. The movement occurred independently of the presence of Z rings and was most easily detectable in cells lacking Z rings. Moreover, we observed oscillation waves of FtsZ-GFP in the helix-like pattern, particularly in elongated cells, and the period of this oscillation was similar to that of the Min proteins. The MreB helix was not required for the rapid movement of FtsZ or the oscillation of MinD. The results suggest that FtsZ not only forms the Z ring but also is part of a highly dynamic, potentially helical cytoskeleton in bacterial cells.

The post-transcriptional processing of tRNAs; decorates them with a number of modified bases important for their biological functions. Queuosine, found in the tRNAs with GUN anticodons (Asp, Asn, His, Tyr), is an extensively modified base whose biosynthetic pathway is still unclear. In this study, it was observed that the tRNA(Tyr) from Escherichia coli B105 (a B strain) migrated faster than that from E. coli CA274 (a K-12 strain) on acid urea gels. The organization of tRNA Tyr genes in E. coli B105 was found to be typical of the B strains. Subsequent analysis of tRNA(Tyr) and tRNA(His) from several strains of E. coli on acid urea gels, and modified base analysis of tRNA preparations enriched for tRNA(Tyr), showed that E. coli B105 lacked queuosine in its tRNAs. However, the lack of queuosine in tRNAs was not a common feature of all E. coli B strains. The tgt and queA genes in B105 were shown to be functional by their ability to complement tgt and queA mutant strains. These observations suggested a block at the step of the biosynthesis of preQ(1) (or preQ(0)) in the B105 strain. Interestingly, a multicopy vector harbouring a functional tgt gene was toxic to E. coli B105 but not to CA274. Also, in mixed cultures, E. coli B105 was readily competed out by the CA274 strain. The importance of these observations and this novel strain (E. coli B105) in unravelling the mechanism of preQ(1) or preQ0 biosynthesis is discussed.

ABSTRACT Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using trmA strains of Escherichia coli(defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.

Formylation of the initiator tRNA is essential for normal growth of Escherichia coli. The initiator tRNA containing the U35A36 mutation (CUA anticodon) initiates from UAG codon. However, an additional mutation at position 72 (72A --> G) renders the tRNA (G72/U35A36) inactive in initiation because it is defective in formylation. In this study, we isolated U1G72/U35A36 tRNA containing a wobble base pair at 1-72 positions as an intragenic suppressor of the G72 mutation. The U1G72/U35A36 tRNA is formylated and participates in initiation. More importantly, we show that the mismatch at 1-72 positions of the initiator tRNA, which was thus far thought to be the hallmark of the resistance of this tRNA against peptidyl-tRNA hydrolase (PTH), is not sufficient. The amino acid attached to the initiator tRNA is also important in conferring protection against PTH. Further, we show that the relative levels of PTH and IF2 influence the path adopted by the initiator tRNAs in protein synthesis. These findings provide an important clue to understand the dual function of the single tRNA(Met) in initiation and elongation, in the mitochondria of various organisms.

Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions, Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world's population and kills about 2-3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli, In this study, use of the yeast two-hybrid assay suggests that the EcoSSB and the MtuSSB are even capable of heterooligomerization. However, the MtuSSB failed to complement a Delta ssb strain of E. coli, The sequence comparison suggested that MtuSSB contained a distinct C-terminal domain, The C-terminal domain of EcoSSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MtuSSB and EcoSSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Delta ssb strain of E,coli, These data allude to the occurrence of a 'cross talk' between the N- and the C-terminal domains of the SSBs for their in vivo function, Further, compared with those of the EcoSSB, the secondary/tertiary interactions within MtuSSB were found to be less susceptible to disruption by guanidinium hydrochloride, Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.