(E)-Methyl 2-[(2-formyl-6-meth­oxy­phen­oxy)meth­yl]-3-phenyl­acrylate

The title compound, C19H18O5, crystallizes with two independent mol­ecules (A and B) in an asymmetric unit in both of which the two aromatic rings are in a bis­ectional orientation as evidenced by the dihedral angle between them [41.7 (1)° in mol­ecule A and 35.6 (1)° in mol­ecule B]. Both mol­ecules adopt an E configuration with respect to the C=C bond. An intra­molecular C—H⋯O hydrogen-bond occurs in mol­ecule A. The crystal packing features inter­molecular C—H⋯O inter­actions

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E.coli,Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show thatE. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli REF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with REF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains betweenmycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with REF arelocated in domains IV and V. those required to specify its interaction with the ribosome are located throughout the molecule. (C) 2010 Elsevier Ltd. All rights reserved

(E)-Methyl 2-[(4-bromo-2-formylphenoxy)methyl]-3-phenylacrylate

The C=C double bond in the title compound, C18H15BrO4, adopts an E configuration. The two rings are almost orthogonal to each other, making a dihedral angle of 82.8 (1)°. An intramolecular C—H...O hydrogen bond occurs. The crystal structure is stabilized by intermolecular C—H...O hydrogen bonds

Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis

The translation elongation factor G (EFG) is encoded by the fusA gene.Several bacteria possess a second fusA-like locus,fusA2 which encodes EFG2. A comparison of EFG and EFG2 from various bacteria reveals that EFG2 preserves domain organization and maintains significant sequence homology with EFG, suggesting that EFG2 may function as an elongation factor. However, with the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like factors has not been investigated. Here, we have characterized EFG2 (MSMEG_6535) from Mycobacterium smegmatis. Expression of EFG2 was detected in stationary phase cultures of M.smegmatis (Msm). Our in vitro studies show that while MsmEFG2 binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of EFGs. Furthermore,unlike MsmEFG (MSMEG_1400), MsmEFG2 failed to rescue an E. coli strain harboring a temperature-sensitive allele of EFG, for its growth at thenon-permissive temperature. Subsequent experiments showed that the fusA2 gene could be disrupted in M. smegmatis mc(2)155 with Kan(R)marker. The M. smegmatis fusA2::kan strain was viable and showed growth kinetics similar to that of the parent strain (wild-type for fusA2).However, in the growth competition assays, the disruption of fusA2 was found to confer a fitness disadvantage to M. smegmatis, raising the possibility that EFG2 is of some physiological relevance to mycobacteria

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli

Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA<SUP>fMet</SUP> wherein the three consecutive G-C base pairs (<SUB>29</SUB>GGG<SUB>31</SUB>:<SUB>39</SUB>CCC<SUB>41</SUB>), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA<SUP>Met</SUP> (<SUB>29</SUB>UCA<SUB>31</SUB>:<SUB>39</SUB>ψ GA<SUB>41</SUB>). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis

Analysis of the fusA2 locus encoding EFG2 in Mycobacterium smegmatis

The translation elongation factor G (EFG) is encoded by the fusA gene. Several bacteria possess a second fusA-like locus, fusA2 which encodes EFG2. A comparison of EFG and EFG2 from various bacteria reveals that EFG2 preserves domain organization and maintains significant sequence homology with EFG, suggesting that EFG2 may function as an elongation factor. However, with the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like factors has not been investigated. Here, we have characterized EFG2 (MSMEG_6535) from Mycobacterium smegmatis. Expression of EFG2 was detected in stationary phase cultures of M. smegmatis (Msm). Our in vitro studies show that while MsmEFG2 binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of EFGs. Furthermore, unlike MsmEFG (MSMEG_1400), MsmEFG2 failed to rescue an E. coli strain harboring a temperature-sensitive allele of EFG, for its growth at the non-permissive temperature. Subsequent experiments showed that the fusA2 gene could be disrupted in M. smegmatis mc<SUP>2</SUP>155 with Kan<SUP>R </SUP>marker. The M. smegmatis fusA2::kan strain was viable and showed growth kinetics similar to that of the parent strain (wild-type for fusA2). However, in the growth competition assays, the disruption of fusA2 was found to confer a fitness disadvantage to M. smegmatis, raising the possibility that EFG2 is of some physiological relevance to mycobacteria