Replacement of the λ boxB RNA–N peptide with heterologous RNA–peptide interactions relaxes the strict spatial requirements for the formation of a transcription anti‐termination complex

Abstract

SummaryIn bacteriophage l, formation of a transcriptional anti-termination complex involving the elongating RNA polymerase is mediated by the interaction of boxB RNA with the RNA-binding domain of the N protein (N peptide). In an attempt to understand the spatial requirements for boxB/N peptide interaction within the anti-termination complex, the effects of changes in the distance between boxA and boxB RNA, the length of the boxB stem, and the distance between the N peptide and remainder of the N protein were examined using a bacterial reporter system. It was found that the requirements for boxB stem length and the distance between N peptide and the remainder of N were optimized and strict. In contrast, replacement of the boxB/N interaction by heterologous RNA-peptide interactions appeared to relax the strict requirement for RNA stem length and the orientation of the RNA-binding peptide, presumably due to the absence of the cooperative interaction between boxB/N and the host factor NusA. In addition, the decrease in activity upon stem lengthening could be partially suppressed by simultaneous lengthening of the RNA spacer. A further understanding of the structural organization of the anti-termination complex may provide insights into how functional ribonucleoprotein complexes may be engineered.

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer-RRE stem II complex

“…DNA cassettes encoding RRE 39 and RRE 68 RNA were cloned into the PstI and BamHI sites of a pACYC394‐based N‐reporter plasmid (pAC nut) upstream of terminator sequences and LacZ, so that binding of the Tat‐derived peptides to the Tat aptamer RNA resulted in antitermination and expression of LacZ. The pBR and pAC plasmids were co‐transformed into Escherichia coli N567 cells (Franklin, ), and β‐galactosidase activity was scored on plates containing X‐gal by comparing the level of blue colony color against a standardized set of controls (RRE‐K1, 6 +; RRE‐RSG‐1.2, 4 +; RRE‐Rev, 3 +; RRE‐RevA20, 2+; RRE‐Tat, 0+) (Sugaya et al , ; Horiya et al , ). Solution assays of β‐galactosidase expression were carried out using ONPG as a chromogenic substrate.…”

RNA‐Directed Amino Acid Coupling as a Model Reaction for Primitive Coded Translation

“…Tat aptamer RNAs were cloned into the PstI and BamHI sites of a pACYC394‐based N‐reporter plasmid (pAC nut) upstream of terminator sequences and LacZ, so that binding of the Tat‐derived peptides to the Tat aptamer RNA resulted in antitermination and expression of LacZ. The pBR and pAC plasmids were co‐transformed into Escherichia coli N567 cells, and β‐galactosidase activity was scored on plates containing X‐gal, by comparing the level of blue colony color against a standardized set of controls (RRE‐K1, 6+; RRE‐RSG‐1.2, 4+; RRE‐Rev, 3+; RRE‐RevA20, 2+; RRE‐Tat, 0+) 16…”

A translational repression reporter assay for the analysis of RNA-binding protein consensus sites

“…(7) The antitermination-based assays are hard to adapt to new protein-RNA interactions due to conformational restrictions and also require very high affinities to produce a read-out. (11,12) The systems based on translational repression require only two designed components and have been demonstrated to work in bacterial, yeast, and mammalian cells. (13)(14)(15)(16)(17) In contrast to the other two systems, the RBP is conformationally free to interact with the target RNA rather than being part of a larger complex making the system more flexible and straightforward to design.…”