doi:10.1038/nindia.2010.180 Published online 29 December 2010

'Promoting' DNA once again

Pawan Kumar Dhar

Is there a way to influence the expression of genes? Is it possible to tweak or synthetically design 'promoters' that are responsible for a particular gene expressing itself in a particular manner?

Manju Bansal

Manju Bansal and her team from Indian Institute of Sciences, Bangalore seem to have found ways to work around this interesting challenge. The team has developed computer programmes that can undertake targeted modification of 'promoter sequences' and engineer new ones thereby opening up a new avenue in controlling gene expression.

What is a promoter?

A cell resembles a 'gated community' of tiny buildings and 'streets'. Keys to enter into any of the tiny buildings are safeguarded in the form of wonderful codes from DNA, encryption of which is much more complex than man-made encryption keys. This DNA is housed in a safe place called the nucleus inside eukaryotic cells.

Detailed study of DNA sequences has revealed families of interesting personalities living under one genomic roof. Some DNA sequences talk a lot, some are reserved, some control the talk of others, while some either appear as artefacts of evolution are on their way to evolutionary retirement.

Interestingly, sequences that talk do not know when to start or stop. The switch that plays a key role in this process is called the "promoter". Special protein molecules called transcription factors bind to promoter sequences and switch 'on' the gene expression on demand. This special binding event also determines how much or how long the talk will last. There's enough scientific evidence to point that gene expression fluctuates even in normal situations.

Unlike electronic circuits, cells use gene expression noise to maintain a robust state many times through degenerate mechanisms. One way to understand this robust behaviour is to collect individual expression data, draw a map of molecular connectivities and study their performance through modelling and simulation. This is the classic systems biology approach.

The other, a minimalist and more recent approach, is to consider individual part behaviour, stitch parts into devices and rationally design devices and networks from scratch. This is popularly called synthetic biology.

Synthetic biology involves construction of 'serene' circuits from an inventory of 'noisy' parts! Since the understanding of individual part behaviour determines the higher-level design of circuits, it is important to experimentally study each part separately and document its quantitative behaviour in a wide range of input conditions. When we focus on parts, the role of promoter sequences emerges as a key requirement to understand individual part behaviour. This is due to the fact that promoter sequences control the on/off state of genes. Thus, for tailoring gene behaviour, it is important to custom-design promoter sequences first.

Design of a promoter is a difficult terrain and depends a lot on the correct fundamental understanding of 'what makes a promoter'.

Clever designs

Bansal says this challenge can be addressed using a series of cleverly designed computational experiments. Her work over the past several years, , , , has led to the identification of key parameters essential for the construction of a promoter.

These parameters include curvature, bendability and stability. By using existing experimental data and computationally applying three key parameters to whole genome sequences, promoter regions were predicted by studying differences between known promoters and non-poromoter sequences.

Simply put, these terms describe the inherent capability of DNA to bend around a given 3 dimensional surface, the extent to which an incoming protein can bend DNA molecule and maximize contact points, and the stability of DNA double helix and DNA-protein interaction. Bansal shows that lower stability of DNA duplex, high curvature and less bendability are key signatures of promoter sequences.

What next?

Chemical synthesis of DNA is fairly routine now-a-days. One can programme machines to churn out long DNA strands, cut and paste them into various combinations and even make a DNA origami in 3D!

However these DNA strands are basically dead on arrival. They do not talk. What gives 'life' to this molecule is the promoter switch that helps photocopy information into RNA molecules and trigger downstream events to keep cells alive. For designing controllable and complex biological circuits from scratch, the ability to build user-defined promoter switches is fundamental to this process.

Bansal and her team have developed a tool called PromPredict that uses a set of free energy threshold values for genomic DNA with varying GC content. The tool shows 99% and 95% sensitivity on E.coli and B.subtilis genomic data and ~ 60 % precision – the highest values obtained so far.

Bansal and her team's work also enables targeted modification of promoter sequences and engineering new ones. "Designing promoters to regulate gene expression is a very challenging task, since the promoter regions often do not adhere to specific sequence patterns or motifs, though some consensus is seen", Bansal says.

"Our promoter prediction algorithm is highly sensitive in identifying promoter regions. It can be used in combination with other physicochemical and structural properties to generate highly accurate predictions."


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