The Ribosome

The ribosome is a complex molecular machine that translates the genetic code into functional polypeptides. We are interested in how this machine catalyzes and coordinates the molecular events of translation and its regulation. Work in the Green lab ranges all the way from translational initiation mechanisms in bacteria to ribosome homeostasis in human disease. In all of our projects, we rely on genetic and biochemical approaches to explore the biology of the system in bacterial, yeast and mammalian systems. Most projects in the lab also incorporate ribosome profiling and other high throughput genome-wide approaches to leverage our biochemical findings into biological insights.

Watch Dr. Green's iBiology.org video explaining Protein Synthesis at https://www.ibiology.org/biochemistry/protein-synthesis/


Ribosomes are RNA and protein machines (PDB 4V88)

Ribosomes are RNA and protein machines (PDB 4V88)

The Lifetime of an mRNA

SunTag runoff experiment. Over time the active ribosomes (green) finish translating, but the mRNA (red) remains. There is a slight offset in the channels for illustration purposes. Data by Dr. Daniel Goldman.

SunTag runoff experiment. Over time the active ribosomes (green) finish translating, but the mRNA (red) remains. There is a slight offset in the channels for illustration purposes. Data by Dr. Daniel Goldman.

In eukaryotes, we follow the steps of mRNA synthesis and processing in the nucleus, translation by the ribosome in the cytoplasm, and mRNA degradation. In bacteria, we similarly explore the interconnections between translation termination and mRNA quality control. Much of our current focus is on the mechanisms of mRNA quality control in eukaryotic systems.

We are using SGA screens in yeast to identify new factors involved in mRNA quality control events. We are using CRISPR screens in mammalian cells to explore stop codon read through and Nonsense-mediated decay (NMD).

We are using microscopy and single mRNA imaging with SunTag to study the molecular details of these and other translation events in real time in living cells.

Revealing Translation Mechanisms in vivo

 

We have developed an improved ribosome footprint profiling methodology that allows us to determine ribosome state at individual codons genome-wide. With this new technology we are exploring the ribosome signals that activate MAPK signaling pathways to regulate translation.

Using ribosome profiling and high-throughput chemical probing we are characterizing the activities of read-through drugs being developed for genetic disorders.

Ribosome states can be distinguished by the length of the mRNA fragments that they protect from RNase.  Wu et al. Molecular Cell 2019

Ribosome states can be distinguished by the length of the mRNA fragments that they protect from RNase. Wu et al. Molecular Cell 2019

 

Biochemistry

 

All of our high-resolution and genetic approaches synergize with in vitro reconstituted systems (bacterial, yeast and mammalian) and with high resolution ribosome profiling.

We are also performing structural studies with cryoEM laboratories to understand all of these events at the molecular level.

Ribosome fractionation on a sucrose gradient. Number of ribosomes in each peak is indicated. Gradient by Dr. Boris Zinshteyn

Ribosome fractionation on a sucrose gradient. Number of ribosomes in each peak is indicated. Gradient by Dr. Boris Zinshteyn

Ribosome Homeostasis

Upsetting the balance of ribosome supply and demand leads to disease.  Mills and Green, Science 2017

Upsetting the balance of ribosome supply and demand leads to disease. Mills and Green, Science 2017

Our interests in ribosome recycling and rescue have raised our interest in ribosome homeostasis and how it contributes to the regulation of gene expression. Why do ribosome deficiencies in different tissues lead to differential sensitivities to these deficiencies? How does the cell balances the demands of protein synthesis with the regulation of initiation, elongation, termination, and recycling steps of this process to ensure that gene expression happens appropriately in order to sustain the life of the organism.