Totally nonlinear, dude!

What’s one of the first thing students learn in high school about nucleic acid molecules? It’s that DNA is double stranded and RNA is single stranded. Not only is this fundamentally incorrect, such type of “linear” thinking is, I would argue, a ‘dangerous’ line of thinking, that may be hard for students to escape from.

Any biochemistry student can tell you that, not only can certain types of RNA form secondary and tertiary structures, but that these structures are critical to understanding the catalytic activity of RNA. This has important revelations for evolution (the RNA-first world hypothesis) and for molecular biology.

Microbiology Bytes ran a segment on The shape of HIV RNA (see image below). The higher order, 3 dimensional structure of HIV RNA could play some important role in viral replication and pathogeneiss

This example serves to illustrate the important point that higher order RNA structure could more important in biology than we could have imagined. Therefore, if we keep giving students the impression that RNA (and other molecules) are linear and static, than they could carry with them a false veiw of biological models.

Coincidently, this also is another reason that computational and mathematical modeling in biology will become all important in the coming years. As it turns out, biochemical structures are just too complex to realistically model without computers (at least not easily).

And, as it turns out, even knowing three dimensional structures may not be enough. Carlos Simmerling from Stony Brook University does computational modeling of biomolecular systems (specifically proteins and nucleic acids) as dynamic entities. In other words, intra-interactions, as predicted by mechanics, cause molecules to “move and shake.” These molecular “vibrations” caused by electrostatic interactions could also greatly potentially affect reactivity of molecules in ways we could have never predicted without computer modeling. Simmerling also develops tools to view these molecules in their energetic states. Here’s a video from Simmerling’s group of the enzyme HIV Protease.

These developments stress the need for new scientists (yes, even biologists) to think mathematically and artistically. We need to be aware of all three dimensions (plus the time dimension) in order to work towards building accurate models of biological and biochemical systems.

Posted in biochemistry, link out, microbiology, musings | 2 Comments »

Intron Splicing

It is well known now that eukaryotic genomes contained gene sequences that are expressed (exons) and intervening sequences that are unexpressed, introns. How are introns removed from the genetic code, and how are intron sequences recognized?

Transcription of DNA occurs wholly. So, when messenger RNA is built by RNA polymerase from the DNA template, the introns are included in this sequence. After mRNA is capped, and elongation of transciption begin intron splicing can occur. After introns have been spliced, the product is mature mRNA, which is then polyadenylated.

Intron-exon junctions studies show that there is a high degree of homology between eukaryotes, the consensus sequence being an invariant GU sequence at the 5′ intron boundary and an AG invariant sequence at the 3′ boundary. These two sequences are all that is required to define a splicing boundary (as we’ll see later).

There are actually 4 different types of introns, but I will focus on the first two types, which work via two general mechanisms, known as Group 1 and Group 2 (creative, I know). Type 1 introns are self splicing and are employed in nuclear, mitochondrial and chloroplast rRNA, tRNA and mRNA. Type 2 introns are also self splicing, and are used in genes in mitochondria and chloroplasts of fungi algae and plants. Type 3 introns require a spliceosome and small nuclear Ribonucleo Proteins (snRNPs) and are responsible for solely eukaryotic splicing. Type 4 introns require ATP and an endonuclease.

As can be seen in the figure, in Type 1 introns, the 3′ OH group of a free guanosine acts a nucleophile, attacking the 5′ phosphate at the splice site, displacing it from the exon. The 3′ OH at the exon, usually a U, acts as a nucleophile, atacking the phosphodiester bond, removing the other end of the intron, and reforming the phosphodiester bond with itself.

In Type II introns, a similar mechanism is used, except that an internal 2′ OH of an adenoside is used as the nucleophile, attacking the 5′ splice site of the exon to produce a lariat structure. In the final step, the 3′ OH of the 5′ exon attacks the phosphodiester bond of 3′ uracil, to remove the lariat.

In type III introns, a lariat structure is formed, but the snRNPs are used to form the secondary structures and to mediate the reaction, though it still occurs without free energy input. There are 6 snRNP subunits that make up the splicesome.

Intron sequences are recognized by short sequence elements within exons, known as exonic sequence/splicing enhancers (ESEs). But the mechanism by which they function is poorly understood. The fact that the splicing process occurs contemporaneously with RNA polymerase transciption elongation allows for the immediate splicing upon the recognition of an exon-intron junction sequence, and may account for the high accuracy of this system. Additionally, some evidence suggests that the C-terminal domain of RNAP II itself helps to tether the exon and splicing machinery together.

Posted in biochemistry | No Comments »