Constructionist Approaches to Biology (Systems Biology) Ananth Grama

Traditional Approaches Cell/Molecular Biology takes a de- constructionist approach Reduce every structure/process/function to its basic blocks and understand these basic blocks first An example of this is the study of individual proteins, binding sites (domains), etc. Much of bioinformatics follows this thought process as well (sequence matching, alignment, motifs, etc.)

Constructionist Approaches Study entire processes and associated system Identify processes Identify constituent elements of processes Quantify flow of information/state The process of studying entire systems with a view to understanding various processes is called Systems Biology.

Understanding Biological Processes The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that information cannot be transferred back from protein to either protein or nucleic acid. replication transcription translation

So how does it all work? DNA codes information Proteins take this information and affect function Proteins (enzymes) are biomolecules that catalyze (i.e., increase the rates of) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products.

So how does it work: Example. β-galactosidase (beta-gal) is a hydrolase enzyme that catalyzes the hydrolysis of β- galactosides into monosaccharides.

So what? Lactose is a β-galactoside Lactose is broken down in the stomach by beta- gal If this does not happen, lactose is not metabilized in the stomach – it makes it to the intestines, where it is broken down by bacteria This causes significant intestinal discomfort (lactose intolerance)

More about beta-gal 1021 Amino acids in E-coli In E. coli, β-galactosidase is produced by activation of the lac operon, as the lacZ gene.

More about beta-gal The lac operon consists of three structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA. lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes β-galactoside permease (LacY), a membrane- bound transport protein that pumps lactose into the cell. lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to beta-gal.

So how does this work? Structure of a gene 5'- XXXXXXXPPPPPXXXXXXPPPPPPXXXXGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGXXXX-3' Gene to be transcribed. (note that the optimal spacing between the -35 and -10 sequences is 17 nt)

Structure of a Gene Prokaryotic promoters In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site. The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes. The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.

So, how does it work? Do we keep producing beta-gal all the time?

So, how does it work? No, we cannot produce all of the proteins involved in all of the processes all of the time! There just isnt the space and the resources in the cell to do this So, what do we do?

So, how does it work? No, we cannot produce all of the proteins involved in all of the processes all of the time! There just isnt the space and the resources in the cell to do this So, what do we do? Only produce it when needed?

So, how does it work? Only produce it when needed? Well, how do I get lactose to control the transcription/translation of the lac operon?

So, how does it work? Only produce it when needed? Well, how do I get lactose to control the transcription/translation of the lac operon?  Perhaps, have lactose bind to DNA and somehow inhibit of enhance the process of transcription/translation?

So, how does it work? Only produce it when needed? Well, how do I get lactose to control the transcription/translation of the lac operon?  Perhaps, have lactose bind to DNA and somehow inhibit of enhance the process of transcription/translation?  No, because if there are a large number of such processes, there just isnt the space and the variability on the DNA.  The cell does this through a process called regulation!

How does regulation work? Let us revisit the lac operon: Notice the gene upstream called LacI?

LacI Gene

LacI LacI generates a protein called lactose repressor in small quantities all the time! In the absence of lactose, the lactose repressor protein binds just downstream of the promoter. This prevents the binding of RNAP, thus inhibiting transcription!

LacI Gene Control When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose, which is a recombination of glucose and galactose, binds to the repressor, causing a change in its shape. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to high levels of the encoded proteins.

Positive Feedback The second control mechanism is a response to glucose, which uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. CAMP binds to the CAP, which in turn allows the CAP to bind to the CAP promoter (on the left in the diagram below), which assists the RNAP in binding to the DNA.

Positive Feedback In the absence of glucose, the prevalence of cAMP and binding of the CAP to the DNA significantly increases the production of β-galactosidase, enabling the cell to digest the lactose needed to produce glucose.

Summary of Positive and Negative Feedback

How do we find these things? Grow cultures on glucose X-Gal is colorless. X-Gal can be cleaved by beta-gal to yield galactose and X (colored) Grow colonies on glucose and X-Gal Grow colonies on lactose and X-Gal What should you expect?