PubMed · 14614835
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David A Shub. 2003-11-11. Q & A.. https://doi.org/10.1016/j.cub.2003.10.041
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T4 RNA ligase 2 (Rnl2) exemplifies a family of RNA-joining enzymes that includes protozoan RNA-editing ligases. Rnl2 efficiently seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA:DNA hybrid but cannot seal DNA nicks. RNA specificity arises from a requirement for at least two ribonucleotides immediately flanking the 3'-OH of the nick; the rest of the nicked duplex can be replaced by DNA. The terminal 2'-OH at the nick is important for the attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester, but dispensable for nick recognition and adenylylation of the 5'-PO4 strand. The penultimate 2'-OH is important for nick recognition. Stable binding of Rnl2 at a nick depends on contacts to both the N-terminal adenylyltransferase domain and its signature C-terminal domain. Nick sensing also requires adenylylation of Rnl2. These results provide insights to the evolution of nucleic acid repair systems.
Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.
Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.