Structural analysis of DNA wrapping in bacterial transcription initiation complex by transmission electron microscopy and single particle analysis

• Autor(a) principal: Ariza, Alfredo Jose Florez
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: http://www.teses.usp.br/teses/disponiveis/76/76132/tde-19102018-090529/
• topic: Análise de partículas isoladas; Complexo da iniciação da transcrição; DNA wrapping; Enovelamento do DNA; Microscopia eletrônica de transmissão; RNA polimerase; RNA polymerase; Single particle analysis; Transcription initiation Complex; Transmission electron Microscopy

The transcription initiation is the first step in gene expression and an important regulation step in all living organisms. In bacteria, it has been proposed that DNA bending and its wrapping on the surface of E. coli RNAP might facilitate the opening of the transcription bubble, which is necessary for the initiation of gene transcription. In this work, it is shown the first structural study to evaluate a DNA wrapping model, including its length and the relative position in the bacterial transcription initiation complex (RP complex), assembled between RNA polymerase-σ70 holoenzyme (RNAP) and a λPR promoter (-100 to +30 wild type). RP complex was prepared and negatively stained with 2% uranyl acetate on a thin-carbon coated grid and the data acquisition of 500 images was performed in a JEM-2100 (JEOL, Japan) microscope equipped with an F-416 CMOS camera (TVIPS, Germany). Single particle analysis of 16,015 particles, grouped in 666 class-averages, was conducted using IMAGIC 4D software (Image Science, Germany) to obtain a three-dimensional model of the RP complex at 20Å resolution. After the rigid-body fitting of the RNAP crystallographic structure (PDB 4YG2) and the modeled DNA promoter, it was observed that the regions 1.2 and 4.2 of the σ70 subunit interacts with the consensus zones, -10 and -35 hexamers of the promoter. Furthermore, it was possible to observe that αCTDs (C-terminal domain) in both alpha subunits would be oriented to facilitate the interaction with the first and second UP-elements regions, respectively (centered around –50 and -75 positions in the promoter). These was enabled by the presence of the characteristics motifs helix-hairpin-helix in these domains. In addition, the downstream DNA, from the transcription bubble, appears to be inside the protein main channel, oriented in a way to enable interactions with the RNAP clamp and jaws. Finally, it was observed that the DNA wrapping has ~32 nm of total length and involves a promoter bent of ~255° around the RNAP surface. The 3D-model obtained in this study is the very first direct structural confirmation of the DNA promoter wrapping in a bacterial transcription initiation complex.