Structural and functional analysis of the NusB-S10 complex shared between transcription and translation
von Xiao Luo
Datum der mündl. Prüfung:2010-05-21
Betreuer:Prof. Dr. Markus Wahl
Gutachter:Prof. Dr. Ralf Ficner
Gutachter:Prof. Dr. Dirk Fasshauer
EnglischTranscription and translation are two highly coupled processes during prokaryotic gene expression where ribosomes initiate translation on mRNAs already during transcription, in contrast to eukaryotes where two principle processes occur in two different cellular compartments. One of mechanisms by which transcription and translation in prokaryotes communicate directly with one other is sharing proteins, which have dual activity. As one such example, S10 protein was initially defined as a ribosomal (r-) protein before an additional role in transcription was discovered. S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. NusB is implicated in translation through studies of its mutations that slow down the translation elongation rate. However, the exact role of NusB in translation remains unknown and the molecular mechanisms by which S10 and NusB can act as transcription or translation factors are still a mystery. Here, regions of S10 dispensable for transcription antitermination were delineated through complementation assays and recombineering. The crystal structure of a transcriptionally active NusB-S10 complex was determined. In the complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive antitermination. Last, the evidence that NusB plays a role of a loading factor in delivering S10 into transcription antitermination complex and into other molecular environment in vitro (crystals) allowed me to deduce a hypothesis that in translation NusB may still function as a loading factor that delivers S10 into ribosomes.
Keywords: antitermination; Crystallography; Ribosomes