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Ribosomes are complex molecular machines that synthesize proteins in all living cells. They translate the genetic message encoded in a messenger RNA (mRNA) to the amino acid sequence of a protein. During this process, ribosomes coordinate several internal conformational changes with the activity of a GTPase elongation factor EF-G to move along the mRNA in steps of one codon (three nucleotides). Folded structures in the mRNA such as hairpins and pseudoknots present mechanical barriers as the mRNA entry tunnel can only accommodate single-stranded RNA. As such, these barriers reduce the elongation rate allowing time for nascent protein folding and, in combination with additional cis elements, can induce ribosomal frame-shifting to synthesize alternate protein products. Despite such a wide-spread effect of mRNA secondary structures on translation, several questions remain unanswered. For example, how do ribosomes coordinate its mRNA unwinding / helicase activity - known to be facilitated by positively charged amino acid residues at the mRNA entry site - with its translocation activity, known to be catalyzed by EF-G? What parts of the translation cycle are affected by the presence of a barrier? Furthermore, how are conformational changes within the ribosome are coordinated with its mechanical movement of the mRNA remains unknown. Single-molecule fluorescence measurements of the ribosome have provided insights into conformational changes of the ribosome and the activity of trans-acting protein factors during translation. However, these studies are blind to the mechanical motion of the ribosome. To this end, optical tweezers have been used to directly measure the movement of the mRNA relative to the ribosome and characterize the effect of mRNA secondary structures on translation. Very recently, a new technology that combines high resolution optical tweezers with single-molecule fluorescence spectroscopy has been developed to monitor multiple reaction coordinates in parallel. This instrument, also known as ‘fleezers’, has made it possible to correlate dynamics within molecules to their mechanical motion along a substrate. In this work, we apply this technique to the complex machinery of translation, the ribosome, to understand how dynamics such as inter-subunit rotation, 30S head rotation and trans-acting factors like elongation factor EF-G, together drive its mechanical movement on the mRNA. Briefly, we establish the functionality of the fleezers by performing tests using a DNA hairpin labeled with FRET probes at its junction. We optimize experimental conditions for two types of measurements, one where a conformational change within the motor is monitored, and other where activity of trans-acting factors is monitored. More broadly, these can be associated to ‘fluorophore on the bead’ and ‘fluorophore in the solution’ type of experiments respectively. We find that in both cases, ideal tether length when using ~1 µm beads is 5 kilobase-pairs. If use of larger beads (~2 µm) is desired, tether length must be increased to at least 8 kilobase-pairs, particularly when the fluorophore is on the bead. The confocal fluorescence detection on the fleezers is limited by the concentration of factor in solution. For the current setup, we find that 10-30 nM of labeled species in solution results in the optimal signal to noise. This concentration range poses a stringent limitation on the type of experiments that can be realistically performed on the fleezers. These optimizations are discussed in chapter 2. We then use this technique to understand how ribosomes unwind downstream secondary structures in the mRNA. We find that unwinding of an mRNA hairpin occurs simultaneously with translocation, and that this process occurs during forward rotation of the 30S-subunit head. Moreover, we modulate the magnitude of the hairpin barrier by force and surprisingly find that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold slower kinetic pathway prior to translocation. This pathway may allow ribosomes to exploit thermal fluctuations to overcome mechanical barriers. We also find that the ribosome occasionally opens the hairpin in two successive sub-codon steps while EF-G remains bound, revealing a previously unobserved translocation intermediate. These results (discussed in chapter 3) motivated us to investigate the role of ribosome conformational changes such as inter-subunit rotation and 30S head rotation in the mechanical movement. In chapter 4, we discuss our preliminary progress towards achieve this goal as well as unforeseen challenges in performing these measurements. In summary, we have established combined force and fluorescence methods in the laboratory and used them to dissect the molecular mechanism of translation. We hope that this work will motivate others in the field to apply similar two-dimensional measurement techniques to understand how molecular motors function.

Ribosomes are complex molecular machines that synthesize proteins in all living cells. They translate the genetic message encoded in a messenger RNA (mRNA) to the amino acid sequence of a protein. During this process, ribosomes coordinate several internal conformational changes with the activity of a GTPase elongation factor EF-G to move along the mRNA in steps of one codon (three nucleotides). Folded structures in the mRNA such as hairpins and pseudoknots present mechanical barriers as the mRNA entry tunnel can only accommodate single-stranded RNA. As such, these barriers reduce the elongation rate allowing time for nascent protein folding and, in combination with additional cis elements, can induce ribosomal frame-shifting to synthesize alternate protein products. Despite such a wide-spread effect of mRNA secondary structures on translation, several questions remain unanswered. For example, how do ribosomes coordinate its mRNA unwinding / helicase activity - known to be facilitated by positively charged amino acid residues at the mRNA entry site - with its translocation activity, known to be catalyzed by EF-G? What parts of the translation cycle are affected by the presence of a barrier? Furthermore, how are conformational changes within the ribosome are coordinated with its mechanical movement of the mRNA remains unknown. Single-molecule fluorescence measurements of the ribosome have provided insights into conformational changes of the ribosome and the activity of trans-acting protein factors during translation. However, these studies are blind to the mechanical motion of the ribosome. To this end, optical tweezers have been used to directly measure the movement of the mRNA relative to the ribosome and characterize the effect of mRNA secondary structures on translation. Very recently, a new technology that combines high resolution optical tweezers with single-molecule fluorescence spectroscopy has been developed to monitor multiple reaction coordinates in parallel. This instrument, also known as ‘fleezers’, has made it possible to correlate dynamics within molecules to their mechanical motion along a substrate. In this work, we apply this technique to the complex machinery of translation, the ribosome, to understand how dynamics such as inter-subunit rotation, 30S head rotation and trans-acting factors like elongation factor EF-G, together drive its mechanical movement on the mRNA. Briefly, we establish the functionality of the fleezers by performing tests using a DNA hairpin labeled with FRET probes at its junction. We optimize experimental conditions for two types of measurements, one where a conformational change within the motor is monitored, and other where activity of trans-acting factors is monitored. More broadly, these can be associated to ‘fluorophore on the bead’ and ‘fluorophore in the solution’ type of experiments respectively. We find that in both cases, ideal tether length when using ~1 µm beads is 5 kilobase-pairs. If use of larger beads (~2 µm) is desired, tether length must be increased to at least 8 kilobase-pairs, particularly when the fluorophore is on the bead. The confocal fluorescence detection on the fleezers is limited by the concentration of factor in solution. For the current setup, we find that 10-30 nM of labeled species in solution results in the optimal signal to noise. This concentration range poses a stringent limitation on the type of experiments that can be realistically performed on the fleezers. These optimizations are discussed in chapter 2. We then use this technique to understand how ribosomes unwind downstream secondary structures in the mRNA. We find that unwinding of an mRNA hairpin occurs simultaneously with translocation, and that this process occurs during forward rotation of the 30S-subunit head. Moreover, we modulate the magnitude of the hairpin barrier by force and surprisingly find that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold slower kinetic pathway prior to translocation. This pathway may allow ribosomes to exploit thermal fluctuations to overcome mechanical barriers. We also find that the ribosome occasionally opens the hairpin in two successive sub-codon steps while EF-G remains bound, revealing a previously unobserved translocation intermediate. These results (discussed in chapter 3) motivated us to investigate the role of ribosome conformational changes such as inter-subunit rotation and 30S head rotation in the mechanical movement. In chapter 4, we discuss our preliminary progress towards achieve this goal as well as unforeseen challenges in performing these measurements. In summary, we have established combined force and fluorescence methods in the laboratory and used them to dissect the molecular mechanism of translation. We hope that this work will motivate others in the field to apply similar two-dimensional measurement techniques to understand how molecular motors function.