20.109(S07): Start-up expression engineering

Introduction

From your work so far this term, you have a good understanding of (at least) two fundamental concepts. From our first experimental module it should be clear that the genetic program for running a cell is readable (through sequencing), writable (through molecular biological techniques and synthesis) and somewhat, though not perfectly understandable. Recall how a genetic part can be as small as 13 base pairs, BBa_B0032 for example, to help carry out the work of protein production by enabling a huge protein/RNA complex, the ribosome, to bind an RNA molecule. From our second experimental module, it should be clear that a cell's programming doesn't end at protein production but rather that proteins are dynamic (chemically and spatially). They react to changes in the envirnoment with great speed and sensitivity. We saw that proteins could be considered "digital" information, either present or absent, but are perhaps better thought of as "tunable" analog data since, as far as the cell is concerned, their relevance is their function, regulated by stability, localization and modification. Thus, from the work we've done so far this term, you may have the idea that gene expression in a cell is powered by the central dogma (DNA making RNA making protein) and then modulated by varying the properties of proteins once they are made. This strategy minimizes reaction times but is, energetically speaking, quite wasteful. Why make a protein if it's not useful? In this experimental module we'll see how nature has refined the genetic programming language so a cell's proteins are not all constitutively expressed but rather are finely regulated at the level of transcription initiation. This turns out to be only one of many points for regulation but it's an important one.

30 nm chromatin fiber, from Robinson et al PNAS 2006

At the same time we'll see how nature has overcome another design issue, namely space constraints. A cell's dimensions do not increase linearly with DNA content. Instead, eukaryotic cells remain compartmentalized and compact the DNA by wrapping it around assemblies of histone proteins called nucleosomes. Nucleosomes wrap around eachother to form chromatin. This solves the space issue, allowing 3 feet of DNA to be crammed into a space perhaps 10 um across, but creates a new problem. Wrapped DNA is less accessible to the transcription and replication machinery. Gene expression becomes newly and intimately related to chromatin dynamics. This new problem is overcome by other multiprotein complexes that interact with the DNA-wrapping proteins. Nucleosomes are redistributed around genes that are "active" though it remains unclear if this redistribution is a cause or a consequence of the activity. We'll study one chromatin-remodeling complex called SAGA in this experimental module.

a model for SAGA's interaction with transcription factors and nucleosome-bound DNA, image courtesy of P. Schultz

The name "SAGA" is an acronym for "Spt-Ada-Gcn5-acetyltransferase." Before describing each of these components, it's important to note that biochemically similar complexes are found in many (all?) of the eukaryotic cells that have been studied. Even more remarkable, these SAGA complexes have similar (identical?) numbers of protein subunits and the proteins have noteable sequence homologies, suggesting conserved functions even in cells with diverse life-styles like yeast and human cells. Natural processes like development and division and disease states like cancer can be understood at the level of transcription (mis)regulation. Chromatin remodeling is required for appropriate gene expression which is, in turn, required for healthy cell behaviors. Thus, there is good reason to believe that an understanding of how SAGA works in yeast can give us insight into its role in cells more medically relevant, like human.

A combination of biochemical and genetic data initially suggested an enzymatic activity, namely a histone-acetyl transferase, encoded by the GCN5 gene in S. cerevisiae existed as a large protein complex called that the authors named "SAGA" [Grant et al 1997].

The yeast we'll be studying, S. cerevisiae, .
SAGA intro 1. Gene expression in euk intimately related to chromatin dynamics 2. SAGA is 19 subunit complex req'd for chrom remodel and appropriate gene expression 3. Only 6 of SAGAs 19 subunits essential for viability. 4. Your mission, should you choose to accept it....

Protocols

Part 3: PCR

1. pRS406 template

DONE!