2024-11-28 ミシガン大学
<関連情報>
- https://news.umich.edu/u-m-multinational-team-of-scientists-reveal-structural-link-for-initiation-of-protein-synthesis-in-bacteria/
- https://www.science.org/doi/10.1126/science.ado8476
細菌のリボソームへのmRNA輸送の分子基盤を解明 Molecular basis of mRNA delivery to the bacterial ribosome
Michael W. Webster, Adrien Chauvier, Huma Rahil, Andrea Graziadei, […], and Albert Weixlbaumer
Science Published:29 Nov 2024
DOI:https://doi.org/10.1126/science.ado8476
Editor’s summary
To express a protein-coding gene, RNA polymerase transcribes DNA into mRNA, and the ribosome subsequently translates the mRNA to a protein. In bacteria, transcription and translation occur simultaneously, and this enables RNA polymerase and the ribosome to cooperate with each other. Using cryo–electron microscopy, Webster et al. visualized the early encounter between a transcribing RNA polymerase and a ribosome, which initiates translation on the nascent mRNA. Supported by single-molecule experiments and in-cell cross-linking mass spectrometry, this study shows how the different elements of the two machineries cooperate to recruit a ribosome to a nascent mRNA. —Di Jiang
Structured Abstract
INTRODUCTION
Genetic information stored in DNA is transcribed into messenger RNAs (mRNAs) by RNA polymerase (RNAP) and translated into protein by the ribosome. In prokaryotes, transcription and translation of a gene occur concurrently and in proximity. This allows RNAP and the ribosome to coordinate their functions. A poorly understood aspect of this coupling between transcription and translation machineries is the potential for RNAP to promote ribosome binding to the mRNA that it is transcribing. A mechanism of mRNA “delivery” to the ribosome would protect the intervening mRNA from ribonucleases, suppress the formation of inhibitory RNA structures, and accelerate transcription by coupling it to the translation activity of the trailing ribosome.
Initial contact between mRNA and the ribosome is generally supported by the ribosomal protein bS1, an RNA-binding protein required for translation of most mRNAs in Escherichia coli. On the ribosome surface, bS1 is located close to the ribosomal RNA sequence that can base pair with a Shine-Dalgarno (SD) motif in the mRNA. Establishment of a stable complex between the ribosome and mRNA may therefore depend on coordination among bS1, the SD motif, and RNAP.
RATIONALE
In the pathway of bacterial translation initiation, the molecular basis of mRNA accommodation in the ribosome and initiator transfer RNA (tRNA) binding have been characterized. By contrast, structural information on the early stages of translation initiation, in which the mRNA and ribosome first interact, is limited. How RNAP contributes to the initiation of translation is also unclear. We sought to visualize how mRNAs are initially engaged by the bacterial ribosome using a combination of structural, biophysical, and proteomic methods.
RESULTS
For structural analysis by cryo–electron microscopy (cryo-EM), we prepared a complex in which the small ribosomal subunit was bound to an mRNA emerging from RNAP. Structures were determined of an ensemble of molecular states that reveal two routes of mRNA delivery from RNAP to the ribosome. In the first, the mRNA emerging from RNAP is bound by ribosomal protein bS1. This sheds light on how bS1 supports initial contact with mRNAs and promotes their unfolding for accommodation within the ribosome. The SD motif of the mRNA is base paired with the ribosomal RNA in an orientation not previously described. The continuous path of the mRNA from bS1 to the site of SD motif recognition reveals how bS1 can deliver mRNA to promote stable ribosome-mRNA complex formation before mRNA accommodation and tRNA recognition.
RNAP was located adjacent to bS1 in these structures. This suggested that bS1 contributes to RNAP-mediated delivery of mRNAs to the pioneering ribosome, and we confirmed this by in vitro single-molecule colocalization experiments. An increased rate of ribosome association to mRNAs within RNAP was observed only in the presence of bS1.
In other structural models, RNAP was tethered to the small ribosomal subunit by the coupling factor NusG. Here, the mRNA was delivered to the mRNA entry channel of the ribosome rather than to bS1. This indicates that NusG and its paralog RfaH likely support an alternative pathway of mRNA delivery to the ribosome. Finally, we confirmed that both contact sites between the ribosome and RNAP observed in our reconstituted sample occur in living cells using in-cell chemical cross-linking combined with mass spectrometry.
CONCLUSION
The structural models provide mechanistic insight into the roles of RNAP, bS1, and the SD motif during the early stages of translation initiation. Supported by kinetic analyses of ribosome binding and in-cell structural proteomic data, we present a model of two pathways of mRNA delivery to the bacterial ribosome.
Early steps in ribosome recruitment to mRNA.
RNAP (gray) synthesizes mRNA (pink) using DNA as a template. The small ribosomal subunit (yellow) is recruited to mRNA through two pathways. In the first, RNAP (green) contacts the ribosomal protein bS1 (cyan), which binds and channels mRNA to the anti-SD motif (top). Alternatively, NusG (teal) tethers RNAP (red) near the ribosomal entry tunnel (bottom). Translation initiation factors promote translation initiation to proceed (not shown).
Abstract
Protein synthesis begins with the formation of a ribosome-messenger RNA (mRNA) complex. In bacteria, the small ribosomal subunit (30S) is recruited to many mRNAs through base pairing with the Shine-Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs, and RNA polymerase (RNAP) can promote the recruitment of the pioneering 30S. Here, we examined 30S recruitment to nascent mRNAs using cryo–electron microscopy, single-molecule fluorescence colocalization, and in-cell cross-linking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S activation. Additionally, bS1 and RNAP stimulate translation initiation. Our work provides a mechanistic framework for how the SD duplex, ribosomal proteins, and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.
