3 research outputs found
Fluorescence-based monitoring of ribosome assembly landscapes
Background Ribosomes and functional complexes of them have been analyzed at
the atomic level. Far less is known about the dynamic assembly and degradation
events that define the half-life of ribosomes and guarantee their quality
control. Results We developed a system that allows visualization of intact
ribosomal subunits and assembly intermediates (i.e. assembly landscapes) by
convenient fluorescence-based analysis. To this end, we labeled the early
assembly ribosomal proteins L1 and S15 with the fluorescent proteins mAzami
green and mCherry, respectively, using chromosomal gene insertion. The
reporter strain harbors fluorescently labeled ribosomal subunits that operate
wild type-like, as shown by biochemical and growth assays. Using genetic and
chemical perturbations by depleting genes encoding the ribosomal proteins L3
and S17, respectively, or using ribosome-targeting antibiotics, we provoked
ribosomal subunit assembly defects. These defects were readily identified by
fluorometric analysis after sucrose density centrifugation in unprecedented
resolution. Conclusion This strategy is useful to monitor and characterize
subunit specific assembly defects caused by ribosome-targeting drugs that are
currently used and to characterize new molecules that affect ribosome assembly
and thereby constitute new classes of antibacterial agents
Validation of a fluorescence-based screening concept to identify ribosome assembly defects in Escherichia coli
NAC controls cotranslational N-terminal methionine excision in eukaryotes
N-terminal methionine excision from newly synthesized proteins, catalyzed cotranslationally by methionine aminopeptidases (METAPs), is an essential and universally conserved process that plays a key role in cell homeostasis and protein biogenesis. However, how METAPs interact with ribosomes and how their cleavage specificity is ensured is unknown. We discovered that in eukaryotes the nascent polypeptide-associated complex (NAC) controls ribosome binding of METAP1. NAC recruits METAP1 using a long, flexible tail and provides a platform for the formation of an active methionine excision complex at the ribosomal tunnel exit. This mode of interaction ensures the efficient excision of methionine from cytosolic proteins, whereas proteins targeted to the endoplasmic reticulum are spared. Our results suggest a broader mechanism for how access of protein biogenesis factors to translating ribosomes is controlled.ISSN:0036-8075ISSN:1095-920