Complex transposons may be replicated by either conservative or replicative transposition.
Some transposons, instead of being cut out, are actually replicated and only the replicated transposon is inserted into a new site.
Process is termed: Replicative Transposition
----- a copy of the transposon still remains in the original location and the overall copy number of the transposon increases in the cell.
Replicative Transposition may involve the formation of a DNA intermediate or an RNA intermediate.
When transposition occurs between two circular DNA molecules an intermediate known as a cointegrate is believed to be formed. In this process, the transposon is replicated and briefly a large cointegrate (which is composed of both circular DNAs and both transposons) is formed by the transposase enzyme. The cointegrate is then resolved by an enzyme known as resolvase into two circular DNAs each containing a copy of the transposon.
Transposition involving RNA intermediates closely resembles the process by which retroviruses copy their RNA genome into a double-stranded DNA molecule using the enzyme reverse transcriptase.
Most transposons found in eukaryotic cells transpose via RNA intermediates including those found in humans.
----- these transposons are first transcribed into RNA.
But in order to integrate into another site of the genome, the RNA must first be transcribed back into DNA.
This occurs via the enzyme reverse transcriptase which is encoded by many transposons. Reverse transcriptase is a kind of DNA polymerase that can use RNA as a template.
(Notice that we have here an exception to the dogma that information will only flow from DNA to RNA to protein. The information (the base sequence) in this case flows from RNA back to DNA.)
Obviously, the movement of a transposon creates mutations in the target site. In the overwhelming majority of cases: mutations will be a disadvantage to the cell.
Example: transposon integrates directly into gene that codes for important protein ----- protein not functional anymore ----- cell may die.
But mutations by transposons are probably important for evolution.
Humans: among others one transposon 6 - 7 kb long called LINE (long interspersed element). Because of many former transpositions ----- occurs in about 50,000 copies in the genome -----
The regulation of eukaryotic gene expression is similar in principle to regulation in prokaryotes. However, in higher eukaryotes such as humans there are more than 200 different types of cells. Each of these cells contains the same genetic information so the genes are differentially expressed in each cell type. There are housekeeping genes which probably represent the majority of the genome, which includes the genes required for general cell structure/function. The housekeeping genes are, therefore, transcriptionally active in all cells.
The regulation of gene expression is primarily at the level of transcription (transcriptional regulation).
As seen in prokaryotes, there are a wide variety of regulatory proteins which bind to specific regulatory sequences on the DNA.
There are cis-acting regulatory sequences [present on the same chromosome and usually adjacent to the actual gene].
promoters
enhancers
GC sequences
silencers
In addition, there may also be trans-acting regulatory sequences [present far from the actual gene being regulated - even on a separate chromosome].
Genes transcribed by RNA polymerase II have 2 core promoter elements:
TATA box
Inr sequence
The promoter may contain a TATA box or an Inr sequence or both.
Recall from lecture on eukaryotic transcription that the TATA box is bound first by TBP (TATA-binding protein), whereas the Inr sequence is usually bound first by a TAF (TATA-binding protein associated factors).
These two sequences are binding sites for the basal transcription factors.
There are other cis-acting regulatory sequences such as the GC box [consensus sequence is GGGCGG]. Specialized transcription factors bind to GC boxes.
In addition, there are commonly sequences known as enhancers.
Enhancers, like promoters, enhance transcription by binding to transcription factors. Enhancers were first described in eukaryotes but are now known to exist in some prokaryotes. The enhancer and the promoter both bind to transcription factors (which bind to each other) and DNA looping results. (see figure in text) This structure is believed to be required for the binding of RNA polymerase.
Gene expression may also be regulated by CHROMATIN STRUCTURE.
Recall that in eukaryotes, DNA is bound to histones in nucleosomal structure. Actively transcribed genes are in the most relaxed form of chromatin (the 10nm chromatin fiber conformation). Nevertheless, a variety of experiments have shown that nucleosomes bound to the promoter or enhancer inhibit the initiation of transcription by blocking the binding of transcription factors and RNA polymerase. Nucleosomal structure is believed to be relieved by proteins known as nucleosome remodeling factors in these regions during transcription. Enhancer and promoter regions of actively transcribed genes contain disrupted nucleosomes or fewer nucleosomes and are, therefore, more susceptible to the action of endonucleases.
Interestingly, the body of transcribed genes remains in nucleosomal structure. Apparently nucleosomes are not an impassable barrier to RNA polymerase. HMG (high mobility group) proteins may disrupt the interaction between the histone proteins (perhaps especially Histone 1) and the nucleosome during transcription.