June's Molecule of the Month: RNA Polymerase, the cell's very own photocopier?

Transfer RNA

We are all (well maybe that's a bit of an exaggeration!) familiar with DNA and genomes, but in the Natural world, RNA is always found, whereas DNA is not! RNA, not DNA is the universal nucleic acid. This is because there are some viruses and bacteriophage that use RNA as the "repository" of their genomic blueprint. Moreover, it is the expression of DNA genomes in the form of RNA transcriptomes that leads to the expression of the phenotype, or characteristics of a cell, and subsequently the organism. Therefore, the central dogma first articulated by Francis Crick: DNA makes RNA makes protein, should be modified to become: "in general, DNA makes RNA makes protein, but sometimes RNA makes DNA, makes RNA makes protein!" The difference between DNA and RNA is subtle. In fact in terms of chemistry, it is simply the presence of an extra oxygen atom on the 2' carbon of the ribose sugar (in DNA, the letter D comes from the deoxy-form of ribose). The presence of this oxygen is sufficient to prevent the formation of a stable B form double helix, without which Watson and Crick would not be household names. It presents a steric barrier to the stable formation of the so called B-form double helix: RNA molecules (like tRNA top left) do often contain double helical regions, but they are usually in short stretches and take the A-form. (Don't worry about As and Bs, they are just historical labels and we also have Z forms which are left handed helices!).

Transcription from DNA 

Many Molecular Biologists would argue that RNA is more important than DNA, but let's just assume both are important. Where does RNA come from? In vivo, DNA and RNA molecules are biopolymers and they are synthesised from a template. That is to say, you need a DNA polymer to make DNA copies in genome replication, and you also need DNA to provide the template for RNA synthesis. (There are some special examples of DNA and RNA synthesis that do not follow this rule, but they wont concern us just yet). Transcription is the name we give to the synthesis of RNA from a DNA template. For example, in order to produce haemoglobin, the organism or cell must first encode the gene (we do, most bacteria have something different). The gene is first prepared for transcription by genome, or more commonly called, chromatin modifying (or remodelling) proteins (imagine unwrapping an orange), the start of transcription often involves a group of protein molecules that mark the start of the gene (the promoter) and these molecules, when in position, provide the impetus for the enzyme (that is the subject of this blog) to bind: RNA polymerase. So technically RNA polymerase is an enzyme that synthesizes RNA from a nucleic acid template using ATP, TTP, UTP and GTP. These NTPs (collective abbreviation) are the building blocks of RNA (recall DNA uses TTP instead of UTP) and their incorporation at the right site is facilitated by the sequence of bases in the template. For example, a DNA sequence (5')GGTTCCAATTTGG(3') would be copied as (5')CCAAAUUGGAACC(3'). (Students, think polarity of synthesis and complementarity of base pairing!)

The enzyme found in E.coli that produces mRNA (and rRNA and tRNA) is simply called RNA Polymerase. In general, one polymerase transcribes all RNA classes in bacteria. In higher organisms, we have an RNA polymerase for each class of RNA: RNA Pol I (rRNA), PolII (mRNA) and PolIII (tRNA). one of the most interesting polymerases is Reverse Transcriptase, which takes RNA templates and converts them to DNA (I will be covering RT as a separate molecule of the month in the future, in view of its special role in AIDS infections).

Eukaryotic RNA Polymerases
comprise multiple subunits

A simple RNA Polymerase will catalyse the polymerisation of RNA, but one of the most interesting features of these molecules (apart from their catalytic mechanism and their level of fidelity), is that they are capable of being regulated in a range of different ways. Organisms from all walks of life are able to activate or repress their RNA Polymerases; or put differently, all organisms can regulate transcription. This may be achieved in one of two generic ways. Firstly, proteins can interact with the core polymerase machinery to change its shape, and as a consequence the catalytic site is either shut down or, in contrast activated. This phenomenon is classically referred to as allostery (from the French for "other site"). In Biology, when an enzyme is modulated in function by the binding of a small molecule, or macromolecule, to a site that is remote from the active site; this is generally a strong indication of regulation. The toggling of enzyme activity in situ, is one of the key mechanisms underpinning the development of complexity in higher organisms (it is also a major "economy saving" device used by cells to control the flux of metabolic intermediates in intermediary metabolism). Allostery goes hand in hand with the second way that RNA Polymerases can be regulated: a phenomenon known as post-translational modification, a topic we shall discuss at a later stage in some detail. However in simple terms, the addition of low molecular weight adduct to a target protein (usually through enzyme-mediated transfer) such as a phosphate or a methyl group, can also induce a shape (conformational) change in the protein, which can indirectly activate or inhibit as with allostery (see the cooperative binding curve for Haemoglobin in an earlier Blog).

Further reading 

Comparisons between the RNA Polymerases in bacteria and animals 

The three eukaryotic RNA polymerases

"On the nature of allosteric transitions: a plausible model": A landmark paper from 1965, in the field of allostery: a substantial paper with a secondary focus on molecular symmetry in proteins. A "must read" for all biochemist. Jacques Monod, Jeffries Wyman and Jean-Pierre Changeux all made major contributions in Science in its widest definition between 1930 and 1990! 

Mark Ptashne: another pioneer in the molecular biology of transcription: from repressors in bacteria to transcription factors in yeast: a remarkable career punctuated by some of the most elegant experiments (he's the accomplished violinist on the left!).Roger Kornberg's lab (awarded the Nobel Prize for determining the structure of RNA Polymerase from yeast): a truly remarkable piece of Biochemistry and X-ray crystallograohy. Robert Tjian: a pioneer in the biochemistry of eukaryotic transcription: I first came across his work in the early '80s along with that of Robert Roeder.