Transcription is the process by which genetic information in the form of DNA is copied into molecules of RNA by the enzyme RNA Polymerase. RNA molecules can either function directly, for example as ribosomal RNA or transfer RNA in the process of protein assembly, or function as messenger RNA that is translated into protein by the ribosome. Transcription takes places in several stages – initiation, in which RNA polymerase binds to a specific sequence in the DNA, melts the DNA hybrid structure and begins synthesis of RNA; elongation, in which the polymerase transcribes the DNA sequence; and termination, in which the polymerase dissociates from the DNA and releases the RNA transcript. All stages of transcription are regulated at multiple levels.

In eukaryotic cells, the structural organization of DNA in the form of chromatin is an important mechanism of transcriptional regulation. In the basic unit of chromatin structure, called a nucleosome, 147 base pairs of DNA are wrapped around an octamer of histone proteins. Nucleosomes generate a strong mechanical barrier to transcription, slowing down transcribing RNA polymerases and in many cases stalling them completely.

In our laboratory we study the effect of nucleosomes on the dynamics of transcription by yeast RNA polymerase II (Pol II). We discovered that the nucleosome slows down elongation and enhances the tendency of Pol II to pause and backtrack. We found that the transcription elongation factors TFIIS and TFIIF enhance the passage of Pol II through the nucleosome by reducing the frequency and length of pauses. By measuring the dynamics of transcription through the nucleosome we were also able to measure the rates of all steps in the elongation cycle of Pol II.

In the cell, DNA is organized in higher order structures of chromatin containing multiple nucleosomes, which are regulated by processes such as chromatin remodeling and post translational modifications of the histone proteins. We are currently expanding the scope of our work to characterize the effect of these factors on transcriptional dynamics.

For more information, see some of our recent papers on transcription:

1. Onoa B, Díaz-Celis C, Cañari-Chumpitaz C, Lee A, & Bustamante C. Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy. ACS Central Science. 21;120(12):e2221309120. (2023)

2. Zhang M, Díaz-Celis C, Onoa B, Cañari-Chumpitaz C, Requejo KI, Liu J, Vien M, Nogales E, Ren G, Bustamante C. Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography. Mol Cell. 82(16):3000-3014.e9 (2022)

3. Díaz-Celis C, Cañari-Chumpitaz C, Sosa RP, Castillo JP, Zhang M, Cheng E, Chen AQ, Vien M, Kim J, Onoa B, Bustamante C. Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products. PNAS. 119(33):e2206513119 (2022)

The ribosome synthesizes proteins along a mRNA template, using a carefully regulated chemical cycle to maintain efficiency and accuracy. During each cycle of amino acid addition, elongation factor G (EF-G) binds the ribosome and hydrolyzes GTP, which allows for the movement of the ribosome one codon along the mRNA in a process called translocation. We are investigating the generation of force during translocation with high-resolution optical traps. Additionally, we are also interested in how the ribosome uses force to overcome translational barriers such as secondary structure.

As the ribosome synthesizes the protein, the nascent chain is threaded through the exit tunnel of the large subunit, which then directs the nascent chain to the solvent. The exit tunnel can sequester about 40 amino acids, and as synthesis proceeds the earlier amino acids are exposed to the cellular environment. This process by its nature means that the N-terminal residues can begin to form structure before the C-terminal residues have been added. Co-translational folding differs from bulk refolding studies due to this sequestering of the chain and also due to steric and electrostatic effects of the ribosome surface. Optical tweezers allow us to probe the folding of the nascent chain with minimal perturbation to the ribosome, so we can study the steps of protein structure formation on the ribosome as well as modulate interactions between the tunnel and the nascent chain.

For more information, see some of our recent papers on translation and protein folding:

1. Alexander L, Goldman D, Wee L, and Bustamante C. Non-equilibrium dynamics of a nascent polypeptide during translation suppress its misfolding. Nature Communications 10 (2019)

2. Desai V, Frank F, Lee A, Righini M, Lancaster L, Noller H, Tinoco I, and Bustamante C. Co-temporal force and fluorescence measurements reveal a ribosomal gear shift mechanism of translation regulation by structured mRNAs. Mol Cell. 75, 1007–1019.e5. (2019)

ClpX is a homohexameric, ring-shaped ATPase that pairs with a barrel-shaped peptidase, ClpP, to form the ClpXP protease complex. ClpX uses the energy from ATP hydrolysis to unfold protein substrates tagged for degradation by threading them into the cavity of ClpP for proteolysis. We studied the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding.

Single-molecule analyses reveal that phosphate release is the force-generating step and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates such as green fluorescent protein. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant “rpm” but uses different “gears.”

For more information, see some of our recent papers on the ClpX motor:

1. Rodriguez-Aliaga P, Ramirez L, Kim F, Bustamante C, Martin A., Substrate-translocating loops regulate mechanochemical coupling and power production in AAA+ protease ClpXP””, Nat. Struct. Mol. Biol. 23 (2016).

2. Sen M, Maillard RA, Nyquist K, Rodriguez-Aliaga P, Pressé S, Martin A, Bustamante C., “The ClpXP protease unfolds substrates using a constant rate of pulling but different gears”, Cell, 155(3), 636-46 (2013).

The φ29 packaging motor is an exceptionally powerful machine that utilizes energy from ATP hydrolysis to translocate DNA into the viral capsid during the last stages of virion assembly. This motor generates forces in excess of 60 pN, overcoming the large energy barriers that resist packaging and compacting a double-stranded DNA genome to near-crystalline density inside the viral head. The motor complex threads the DNA through its central pore and consists of three coaxial rings: a dodecameric connector, a pentameric pRNA ring, and a homo-pentameric ring, of gene product 16 (gp16), the ATPase that constitutes the catalytic core of the motor.

Over the past decade, we have completed a series of single-molecule experiments that have elucidated many aspects of the mechanochemical conversion process of viral genome packaging. The dsDNA bacteriophages, such as φ29, serve as excellent models for the medically relevant herpes- and adenoviruses since they share a similar packaging mechanism.

ASCE ring ATPases are thought to employ a highly conserved set of structural elements for intersubunit coordination, yet they display varying modes of motor coordination. However, little is known about how the information needed to maintain the timing and coordination of these transitions propagate around the ring and what interactions are responsible for this propagation. We investigate the molecular mechanisms responsible for the intersubunit communication and coordination in the φ29 motor.

Motor-substrate interactions can be specific or nonspecific in nature.  Since φ29 has a well-characterized biphasic “dwell-burst” mechanism that employs both types of contacts, it is an ideal model to investigate how motor-substrate interactions impact motor regulation and force generation. Moreover, almost every aspect of the mechanics of the φ29 packaging motor operation responds dramatically to the amount of DNA packed inside the capsid. Extensive alterations in mechanics reflect an allosteric mechanism that is likely communicated from the capsid to the ATPase. We also investigate how the φ29 packaging motor, and possibly a broad range of force-generating motors, can have its fundamental mechanics regulated by such an allosteric mechanism.

For more information, see some of our past papers on the φ29 packaging motor:

1. Liu S, Chistol G, Hetherington CL, Tafoya S, Aathavan K, Schnitzbauer J, Grimes S, Jardine PJ, Bustamante C. A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills
Cell, Vol. 157 pp.702-713, (2014)
2. Chistol G, Liu S, Hetherington CL, Moffitt JR, Grimes S, Jardine PJ, Bustamante C. High Degree of Coordination and Division of Labor Among Subunits in a Homomeric Ring ATPase
Cell, November 21, Vol. 151, no. 5, 1017-1028

Enzymes catalyze chemical reactions. They are an essential element of the biological realm because if all reactions occurred without enzymes, the reaction would be so slow that life could not be sustained. It is known that enzymes increase chemical reaction rates by lowering the activation energy of those reactions and, since the pioneering studies of Michaelis and Menten, have been assumed to remain unaltered during the process. The heat released during a chemical reaction is much greater than the energies maintaining the stability of the catalyst itself.

Recently it was found that the diffusion coefficient of enzymes, as measured in Fluorescence Correlation Spectroscopy (FCS) experiments, increases significantly in the presence of their substrate (Muddana, H. S. et al., JACS,132(7), 2110 (2010)). The authors formulated an ‘electrophoretic’ model as well as a ‘chemotactic’ one to explain this observation. Using different enzymes, we have shown that the increase in diffusion coefficient is related the amount of heat released by the reaction during each turnover. A key point of this study is that enzymes with large enthalpy exhibit a high enhanced diffusion while enzymes with nearly null enthalpy do not. According to our model, the reaction produces a heat wave that propagates from the active site of the enzyme, deforming it against the protein-surface interface, giving rise to a recoil effect that effectively propels the enzyme during each catalytic event. We formulate a stochastic theory that predicts the increase in diffusion coefficient to be proportional to the rate of the reaction, as is indeed observed experimentally. This formulation also shows that the coefficient of proportionality depends linearly on the enthalpy of the reaction.

Our results suggest a crucial re-thinking of the current paradigm of enzyme catalysis: with the energy released easily one order of magnitude larger than the free energy stabilizing the protein catalyst, it is not unthinkable that many enzymes may partly unfold after each catalytic event and that their turnover measured in bulk may include a ‘dead time’ while the enzyme regains its active structure. Similarly, we speculate that some enzymes, particularly those that function as molecular motors, may have evolved to ‘channel’ the energy released after each catalytic event to help propel them along their tracks.

We believe that this work should be of interest to a broad range of scientists, including enzymologists, biochemists, biophysicists, and in general, those interested in energy exchange and energy flow phenomena at the nano scale.

For more information, see our past paper:

Riedel C, Gabizon R, Wilson CAM, Hamadani K, Tsekouras K, Marqusee S, Pressé S, and Bustamante CJ, “The heat released during catalytic turnover enhances the enzymes diffusion”, Nature 517, 227–230 (2015).