Periodic Reporting for period 3 - SMACK (Single-molecule tracking for live-cell protein synthesis kinetics)
Reporting period: 2024-01-01 to 2025-06-30
Ribosome-catalyzed synthesis of proteins according to the genetic information stored as DNA is fundamental to all living cells. Proteins perform practically all the daily work of a cell, such as transporting molecules from one part of the cell to another and, particularly, catalyzing and regulating all chemical reactions taking place in the cell. The molecular machinery of protein synthesis has been characterized in great detail, mainly using reconstituted systems where the individual components have been purified from cells and then studied in the test tube. However, whereas powerful to dig out the details, this reductionist approach can never account for all the aspects of the complex, crowded environment of the living cell. The aim of this project is to fit the detailed pieces of information on the process of protein synthesis into a bigger picture taking into consideration the full plethora of players present in the cell. In particular, we want to understand how the sequence of the template, the mRNA, determines how, when, and where the different proteins should be folded, and, in particular, how these decisions are made during ongoing protein synthesis. This information will be particularly important for optimization of protein production, such as cost-effective production of therapeutic proteins using bacterial cells. To achieve this, we are developing fluorescence-based tools to study individual molecules, one by one, directly during action, inside living E. coli cells.
The project is divided into three main subprojects:
• Subproject 1: How are mRNA translation rates tuned, globally and locally, to allow for rapid polypeptide production, whilst still maintaining proper polypeptide folding and/or targeting to non-cytosolic compartments?
• Subproject 2: What is the timing and capacity of Signal Recognition Particle (SRP)-mediated cotranslational targeting of nascent polypeptides to the membrane-bound peptide translocation complexes (i.e. the translocons)?
• Subproject 3: How are mRNA translation rates affected, globally and locally, by ribosome-targeting antibiotic drugs, and how is this connected to the drug’s bacteriostatic/bactericidal effect?
The project is divided into three main subprojects:
• Subproject 1: How are mRNA translation rates tuned, globally and locally, to allow for rapid polypeptide production, whilst still maintaining proper polypeptide folding and/or targeting to non-cytosolic compartments?
• Subproject 2: What is the timing and capacity of Signal Recognition Particle (SRP)-mediated cotranslational targeting of nascent polypeptides to the membrane-bound peptide translocation complexes (i.e. the translocons)?
• Subproject 3: How are mRNA translation rates affected, globally and locally, by ribosome-targeting antibiotic drugs, and how is this connected to the drug’s bacteriostatic/bactericidal effect?
Subproject 1 - We have completed and published an initial proof-of-concept single-molecule tracking study of ribosomal subunits in living E. coli cells (Metelev et al 2022 Nat Commun). Due to the significantly improved tracking quality achieved with our labelling approach, we were able to deduce hitherto unattainable kinetics data of mRNA translation initiation and elongation. Our most striking finding was that re-initiation on poly-cistronic mRNAs, without ribosome dissociation, probably occurs by complete 70S ribosomes, and not by only the small 30S subunit as has been commonly believed. In the article, we also present results from our attempts to track orthogonal ribosomes, with which we had planned to measure mRNA-specific translation rates. It turned out that the Shine-Dalgarno (SD) – anti-Shine-Dalgarno (anti-SD) interaction between the mRNA and small ribosomal subunit is not enough to direct translation initiation in E. coli, but that other factors contribute significantly to the selectivity of this process. Hence, we have so far not been able to set up an experimental system to reach the goals of subproject 1, but are working on other solutions.
Subproject 2 – We have reported an initial study of SRP tracking, in which we present a kinetic model for SRP-mediated co-translational targeting of polypeptides to the translocons in the cell membrane (Volkov et al. 2022 PNAS). Our results suggest that: (i) SRP do not bind stably to non-target ribosomes, i.e. ribosome sampling must be faster than the time resolution of our experiments (< 40 ms); (ii) the SRP-ribosome complex finds a vacant translocon through a combination of 3D and 2D search, where the SRP receptor (FtsY in E. coli) aids in anchoring the complex to the inner membrane; and finally (iii) that SRP-mediated targeting is very fast (<1 s on average) which explains why SRP-mediated ribosome stalling is not needed in a small bacterium such as E. coli. Currently, we are following up on the study, in accordance with the proposed plan, by developing experimental systems to track other components of the pathway and competing pathways, such as the SRP receptor FtsY and translocons, as well as the ribosome-associated chaperone Trigger factor. Due to intrinsic difficulties in analysing 2D trajectories of membrane-bound components, we are also extending and improving our optical system to be able to track fluorescently labelled molecules in 3D.
Subproject 3 – We investigated the effects of the antibiotic compound kasugamycin on translation in our ribosome tracking article (Metelev et al 2022 Nat Commun). We are currently optimizing a microfluidics system with which we will be able to deliver antibiotics to cells in a synchronized manner, and follow the effect on the cell physiology and protein synthesis machinery (using e.g. labelled ribosomes) on a sub-minute timescale.
Subproject 2 – We have reported an initial study of SRP tracking, in which we present a kinetic model for SRP-mediated co-translational targeting of polypeptides to the translocons in the cell membrane (Volkov et al. 2022 PNAS). Our results suggest that: (i) SRP do not bind stably to non-target ribosomes, i.e. ribosome sampling must be faster than the time resolution of our experiments (< 40 ms); (ii) the SRP-ribosome complex finds a vacant translocon through a combination of 3D and 2D search, where the SRP receptor (FtsY in E. coli) aids in anchoring the complex to the inner membrane; and finally (iii) that SRP-mediated targeting is very fast (<1 s on average) which explains why SRP-mediated ribosome stalling is not needed in a small bacterium such as E. coli. Currently, we are following up on the study, in accordance with the proposed plan, by developing experimental systems to track other components of the pathway and competing pathways, such as the SRP receptor FtsY and translocons, as well as the ribosome-associated chaperone Trigger factor. Due to intrinsic difficulties in analysing 2D trajectories of membrane-bound components, we are also extending and improving our optical system to be able to track fluorescently labelled molecules in 3D.
Subproject 3 – We investigated the effects of the antibiotic compound kasugamycin on translation in our ribosome tracking article (Metelev et al 2022 Nat Commun). We are currently optimizing a microfluidics system with which we will be able to deliver antibiotics to cells in a synchronized manner, and follow the effect on the cell physiology and protein synthesis machinery (using e.g. labelled ribosomes) on a sub-minute timescale.
Our most significant achievements lie in the combination and optimization of cutting-edge experimental and analytical techniques. In particular, the combination of new labeling strategies and careful benchmarking of biomolecules labelled with new brighter and more photostable dyes, with our continuously improved and expanded machine-learning-based analysis pipeline, supplemented with state-of-the-art microscopy simulations, have allowed us to disentangle and quantify biochemical pathways inside living cells at an unprecedented level (Volkov et al 2022 and Metelev et al 2022).
We are currently finalizing a study of the ribosome-associated chaperone Trigger factor (TF). Before the end of the project, we should have a complete picture of TF’s ribosome binding dynamics, including delineation of a potential competition between SRP and TF for ribosome binding.
We are improving our instrumentation and analysis pipeline both in order to track molecules in 3D, as well as to study interactions using FRET. With these enhancements, we will hopefully be able to dissect in even greater detail the binding mode and order of events regarding SRP’s and SRP receptor’s association to ribosomes, the membrane, and ultimately the translocons.
Since our initial design of orthogonal ribosomes, utilizing altered SD-anti-SD sequences has proven not to work, we are currently investigating other possibilities to connect or direct a ribosome to a specific mRNA. One of the major challenges is to keep the engineered system as functional and natural as possible, to be able to still study relevant biology.
We are currently finalizing a study of the ribosome-associated chaperone Trigger factor (TF). Before the end of the project, we should have a complete picture of TF’s ribosome binding dynamics, including delineation of a potential competition between SRP and TF for ribosome binding.
We are improving our instrumentation and analysis pipeline both in order to track molecules in 3D, as well as to study interactions using FRET. With these enhancements, we will hopefully be able to dissect in even greater detail the binding mode and order of events regarding SRP’s and SRP receptor’s association to ribosomes, the membrane, and ultimately the translocons.
Since our initial design of orthogonal ribosomes, utilizing altered SD-anti-SD sequences has proven not to work, we are currently investigating other possibilities to connect or direct a ribosome to a specific mRNA. One of the major challenges is to keep the engineered system as functional and natural as possible, to be able to still study relevant biology.