Chromatin remodeling of nucleosomes
Fig. 13.7 Control of eukaryotic transcription. (a) The availability of the transcription template is controlled by chromatin structure. This is generally a developmental process and condensation of chromatin into heterochromatin is often essentially irreversible in a differentiated cell. Such condensed chromatin is completely transcriptional silent and contains unacetylated histones and methylated DNA. Euchromatin is more loosely organized with acetylated histones; in such regions genes are essentially off, but some transcription can occur at irregular intervals or during chromosome replication. Activation of genes in euchromatin as a result of the binding of transcriptional activators to regulatory sequences such as enhancers leads to high levels of transcription. (b) The JakSTAT signal transduction cascade induced by a interferon resulting in transcriptional activation of interferon induced genes. As discussed in the text, the presence of a interferon leads to dimerization of specific receptors at the cell surface leading first to the phosphorylation of Jak bound to the C-terminal cytoplasmic domains of these receptors followed by the phosphorylation of the receptor peptides themselves. This results in the phosphorylation of cytoplasmic STAT proteins, which dimerize and migrate to the nucleus where they activate appropriate transcription.
presence of interferon leads to the formation of a heterodimer on the cell surface. Each of the subunits has a Jak protein associated with it, and the dimerization allows these proteins to phosphorylate each other — the phosphorylated Jak proteins then phosphorylate receptors on the C-terminal regions of the a interferon receptors themselves. These in turn serve as sites for the association of STAT proteins with the complex, and the Jaks phosphorylate them leading to their disassociation from the complex, dimerization, and migration to the nucleus where they bind to regulatory sequences of a interferon inducible transcripts such as those encoding 2',5-oligoA synthetase, PKR, etc. Thus, a single signal outside the cell can lead to multiple responses (a cascade) directed at that signal.
Following initiation of transcription, transcript elongation proceeds. RNA is also modified following initiation by addition of a cap at the 5' end. Capping takes place by addition of a 7-methyl guanine nucleotide in a 5'-5' phosphodiester bond to the first base of the transcript. This cap has an important role in initiation of protein synthesis. Transcription proceeds until the pol II—nascent transcript complex encounters a region of DNA-containing sequences pro-
IFN receptor monomer
Cross phosphorylation of JAKs
IFN receptor dimer
IFN receptor dimer
Phosphorylation of C-terminal portion of receptors c>
STATS dock on receptor
Phosphorylation of STATS
Migration to nucleus and activation of IFN response genes
Dissociation from receptor and dimerization of phosphorylated STATS
Fig. 13.7 Continued viding transcription-termination/polyadenylation signals that occur over 25—100 base pairs. A major feature of this region is the presence of one or more polyadenylation signals, AATAAA in the mRNA sense strand.
Other short cis-acting signals also are present in the polyadenylation region. A specific enzyme (polyA polymerase) adds a large number of adenine nucleotides at the 3' end of the RNA just downstream (3') of the polyadenylation signal as it is cleaved and released from the DNA template. Interestingly, the polymerase itself can continue down the template for a short or a long distance before it finally disassociates and falls off.
In addition to capping and polyadenylation, most eukaryotic mRNAs are spliced. In splicing, internal sequences (introns) are removed and the remaining portions of the mRNA (exons) are religated. Splicing takes place via the action of small nuclear RNA (snRNA) in complexes of RNA and protein (ribonucleoprotein) called spliceosomes. The process is complex, but the result is that most mature eukaryotic mRNAs are somewhat or very much smaller than the pre-mRNA precursor or primary transcript.
The generation of mature mRNA in the nucleus is shown diagrammatically in Fig. 13.8. Although splicing is shown to occur after cleavage/polyadenylation in this diagram, the actual process may occur as the nascent RNA chain grows. The maturation of RNA and splicing are shown in a somewhat higher-resolution view in Fig. 13.9.
All modifications occur on the RNA itself: first capping, then cleavage/polyadenylation of the growing RNA chain, then splicing (if any). Thus, almost all eukaryotic mRNAs are capped,
6. Translation of protein
polyadenylated, and spliced. Because splicing can occur within or between sequences of mRNA encoding peptides, it can result in the generation of complex "families" of mRNA encoding related or totally unrelated proteins. Some general patterns of splicing known to be important in virus replication are shown in Fig. 13.10(a).
Visualization and location of splices in eukaryotic transcripts
Provided a good physical map and cloned copies of the eukaryotic gene encoding a spliced transcript are available, there are a number of techniques for detecting and locating the splice sites in a given transcript; three are shown in Fig. 13.10(b). All are based on the fact that when the DNA gene is hybridized to the mature transcript, the introns present in the gene will not be able to hybridize and, therefore, must form a single-stranded loop in an otherwise contiguous hybrid.
The unhybridized ssDNA loop can be visualized in the electron microscope using a technique called R-loop mapping. R-loop mapping was originally developed as a method of visualizing a DNA—RNA hybrid in a dsDNA molecule by allowing hybridization under
A "typical" transcription unit
5' splice site 3' splice site mRNA sense strand | |
DNA sense 5'|tatat 20 bases a 150 bases aagatggtc 150 bases aggtgagt 300 bases ctcaccaggt 450 bases taa 25 bases aataaa 3'
DNA antisense 3' |atata 20 bases t 150 bases ttctaccac 150 bases tccactca 300 bases gagtggtcca 450 bases att 25 bases ttattt 5' mRNA template
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