Structural and Functional Analysis of the E. coli NusB-S10 Transcription Antitermination Complex

Abstract

Summary Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this 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.

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

“…In addition, we also took an unbiased approach using UV-cross-linking followed by MS to identify protein-RNA contact sites (Luo et al 2008;Kramer et al 2011). …”

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…It was equipped with four 8 W lamps (dimensions 1.5 cm  28.5 cm; wavelength 254 nm; G8T5, Sankyo Denki, Japan) as described elsewhere [32]. Alternatively, a UV stratalinker 2400 from Stratagene can be used.…”

“…2. The sample is transferred to a black polypropylene microtiter plate (Greiner Bio-One); aliquots of 100 ll are placed in each well, and the plate is placed on an ice-cold metal (aluminum) block (see [32] for details). 3.…”

Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis

“…S10 is also known to act as a transcriptional antitermination factor (Friedman et al, 1981;Squires and Zaporojets, 2000). The S10-NusB complex is associated with the BoxA sequence of rRNA being transcribed and participates in processive transcription antitermination of rRNA operons in E. coli (Luo et al, 2008;Quan et al, 2005). When the number of intracellular S10 proteins exceeds that of ribosomes, the excess S10 promotes rRNA transcription, thus allowing positive feedback control of ribosome synthesis (Luo et al, 2008).…”

“…The S10-NusB complex is associated with the BoxA sequence of rRNA being transcribed and participates in processive transcription antitermination of rRNA operons in E. coli (Luo et al, 2008;Quan et al, 2005). When the number of intracellular S10 proteins exceeds that of ribosomes, the excess S10 promotes rRNA transcription, thus allowing positive feedback control of ribosome synthesis (Luo et al, 2008). In B. subtilis, it has been reported that the ratio of NusA, a well-investigated anti-termination factor, to RNA polymerase in rRNA-transcription elongation complexes is 2 1, and that S10 (NusE) and other anti-termination factors are required to recruit a second molecule of NusA to the rRNA elongation complex .…”