Uncontrolled transposition is deleterious to the cell and thus, the frequency of transposition is generally kept to a minimum by various regulatory mechanisms. Describe the FOUR ways that transposition is controlled. Transposition is the movement of a particular fragment of DNA from one part of a genome to another.
A transposon is a segment of DNA which is capable of moving from a specific location on a DNA molecule to another location on the same or different molecule. For this reason, it is known as a “jumping element”. The recombination that takes place involves two unrelated sequences.This is unlike other homologous recombination events such as crossovers in meiosis and in transposition; there is a completely new arrangement of genes along the chromosome. A transposon could contain antibiotic resistance genes so that when it inserts itself into its target, it could confer resistance to the host. Transposons have therefore aided the development of plasmids which give multiple drug resistance to certain bacteria. Transposition can be both beneficial and hazardous to the host.
Over time, transposons have led to genetic variability and evolution.This is due to their ability to generate mutations by insertion within a host’s genome. However, their insertion can lead to alterations in DNA arrangement such as cause deletions, inversions and chromosome fusions. For this reason, transposition can be deleterious. It is important to understand how the activity of transposable elements is regulated. Transposition activity must be limited so that there is little capacity to damage host DNA but still maintain advantageous features. For this to be achieved, a balance must be struck between too much transposition occurring and too little.
This is known as the frequency of transposition. This essay will review the different types of regulatory mechanisms employed. A transposon element consists of three major regions. It contains a gene for transposase, insertion sequences (IS) and a coding region for proteins such as those which give antibiotic resistance. These multiple protein-coding regions lie in between the short, repeated sequences. Transposase employs the joining together of the transposon to the host’s genome through a cut and paste mechanism whereby the enzyme cleaves its transposon and splices its ends to the target sequence.This is known as conservative transposition.
The insertion sequences can be direct or inverted repeats of DNA. Directionality is given for being on different ends of a transposon. Transposition occurs because of the insertion sequences in the terminals of the transposon and the transposase enzyme . Composite transposons are similar to simple transposons. Tn5 is an example of a composite transposon because it is flanked by two separate IS elements. Its structure is shown below in figure 1: Figure 1: Tn5 Transposon, adapted from Annual Review Microbiology, 47: 945-63, Reznikoff, 1993The Tn5 encodes two proteins, the transposase enzyme with a related protein and the transposition inhibitor. Since transposons are defined by the specific sequence at its ends, changes in any base pair within these sequences can typically reduce the frequency of and in some cases, completely inhibit transposition.
The abundance of the inhibitor is one means of determining the frequency of its transposition. The synthesis of the two proteins that Tn5 codes for is regulated by a set of genetic regulatory elements.The proteins that the host encodes also play a crucial role in the transposition process. The host DNA methylation function also plays a part in controlling transposition because the expression of transposase is sensitive to DNA methylation. The transposase enzyme is highly unstable and in any case, cannot accumulate to very high levels in the cell. This in itself is a self-regulatory mechanism. The expression of transposase is also controlled during translation.
This is done by blocking ribosome docking so that transposase translation is minimised.This is mainly seen to occur in Tn10 however. There are therefore many ways in regulating transposition. Most of the above regulatory mechanisms occur in Tn5 except at the translation level, which has seen to occur in Tn10 (Reznikoff, 1993). Mobility of bacterial transposons is usually regulated to approximately 10-3 to 10-8 transpositions per element per bacterial generation (Horak & Kivisaar, 1999). The main factor affecting the rate of transposition is the amount of active transposase enzyme available.Without transposase, the transposon would not be able to insert itself into a host’s genome.
The mechanisms described briefly above are down-regulatory and must operate frequently at different points of transposase expression to ensure precise control. In the absence of any control mechanisms, the number of transposon copies per cell would increase continuously over time. There are therefore means by which the rate of transposition per copy is reduced as the number of transposon copies rises. The Tn5 contains two nearly identical sequences, IS50R and IS50L.The IS50R is a fully functional transposable element coding for the transposase (Tnp), but the IS50L contains a codon which results in the synthesis of inactive proteins. Therefore, the transposon has two opposing activities. In this way, the Tnp can inhibit the activity of other Tnp molecules.
The Tnp, binds to the OE and IE end sequences (see diagram). The IS50R also encodes another protein and this is the inhibitor (Inh). The expression of this inhibitor causes it to bind to the transposase molecule and prevent post-cleavage events. Unlike Tnp, the Inh lacks the N-terminal 55 amino acids.Its function in trans is to inhibit transposition. As well as the activities of both Tnp and Inh, their relative abundance plays a major role in regulating the frequency of transposition (Sasakawa et al, 1982). As the number of Tn5 increases, the concentration of trans-acting Inh increases so as to stop further transposition.
However, the amount of cis-acting Tnp remains constant. Transposase is preferentially cis acting. It is now known that there are promoters which program Tnp and Inh syntheses. There is also regulation of these key promoters. The level of transposase is limited primarily by gene translation.This has seen to occur in IS10, IS50, Tn3 and IS903. The translation of mRNAS of the transposases of the IS10 and IS30 regions are inhibited by anti-sense RNAs.
This RNA is encoded from a promoter that generates a transcript called “RNA-OUT” which is complementary to the 5’ end of the transposase mRNA (Nagy & Chandler, 2004). The anti-sense RNA pairs with the 5’ end of this mRNA and this blocks ribosome binding. Therefore, translation cannot occur. The anti-sense RNA is a stem-loop structure and pairing is initiated by an interaction between G and C residues at the 5’ end of the mRNA and the top of the loop.Since the anti-sense RNA is tenfold more abundant than the mRNA of transposase, it is more stable so that transposase translation is greatly reduced. In IS10, translation is further reduced due to fold-back inhibition. This is when a region of transposase mRNA pairs with and stops the functioning of the upstream ribosome binding site (Kleckner, 1990).
Dam DNA methylation down-regulates the synthesis of Tnp. The bacterial DNA adenine methyltransferase is a product of the dam gene which methylates the N-6 position of adenine on both strands of the DNA symmetrical sequence.IS10, IS50 and IS903 carry the GATC methylation sites in their transposase promoter regions. By methylating these sites, the transposase is inhibited from binding to the IE. A fully methylated transposase promoter does not allow the binding of RNA polymerase yet a hemi-methylated promoter permits this binding. Hemimethylated DNA is generated whenever a methylated GATC site is replicated. The GATC sites are placed strategically in the transposase gene promoter region at one end of the element and in the transposase binding site at the other.
Therefore, methylation at these locations affects both transposase gene expression and the transposition process. When these sites hemimethylated, there is an increase in activity of the promoters and hence transposition (Kleckner, 1990). The means by which transposition is controlled were packaged into four fundamental methods. These were the effects of dam methylation, translational regulation, transposase inhibitors and the way in which transposase may be able to self-regulate. There are many more ways in which transposition is regulated.Most of the regulations described above are due to controlling the level of transposase expression as this is the enzyme which allows the transposition mechanics to occur. Other means of regulation are due to the intervention of host protein factors.
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