Transcript Trnascription in eucaryotes

Transcription in eucaryotes
The basic chemistry of RNA synthesis in eukaryotes is
the same as in prokaryotes.
Genes coding for proteins are coded for by RNA
polymerase II, which is a complex enzyme.
Capping the RNA transcribed by
RNA polymerase II
•
•
•
During the initial synthesis of an mRNA the first
NTP retains its triphosphate group.
Capping consists of removing the  phosphate
and adding a GMP 5´-5´ (Fig. 24.11). The G
which is added is then methylated at N-7 and
the ribose of the original G is methylated on the
2´-OH. A further methyl may be added to the
next ribose.
Capping protects mRNA from exonucleases
and is involved in translation initiation.
Split genes
Split genes
•
•
•
In eukaryotes genes contain non-protein coding
regions called introns that interrupt the coding
regions.
A gene can contain as many as 500 introns that
vary from 50-20,000 base pairs in length.
The primary transcript must be edited to
remove the introns before translation can occur.
Mechanism of splicing (1)
•
•
•
•
The key reaction in splicing is transesterification, where a phosphodiester bond is
transferred to another OH group, breaking the
RNA chain without any loss of energy.
Introns always begin with GU and end with AG.
The actual splicing reaction is catalysed by
spliceosomes, which contain small nuclear
RNA.
SnRNAs are associated with proteins to form
small ribonucleoprotein particles (snRNPs). A
spiceosome contains about 300 different
proteins and small nuclear RNAs U1, 2, 4, 5
and 6.
Mechanism of splicing
Ribozymes and the self-splicing
of RNA
• This was first shown to occur in rRNA from
Tetrahymena and to require a divalent metal ion
and guanosine but no protein (Fig. 24.14).
• Initially the action was cis but subsequently
ribonuclease P was shown to be a trans acting
ribozyme.
What is the biological status of
introns?
•
•
•
In evolution introns may have accelerated the rate
by allowing exon shuffling.
Since exons often correspond to protein domains
this is akin to developing a new piece of electronic
equipment by rearranging whole boards rather
than individual transistors.
Introns facilitate shuffling because the exons must
be retained intact during chromosome
rearrangement whereas rearrangements which
change introns are not likely to be harmful since
they have no function.
What is the origin of split genes?
(1)
•
•
One view is that prokaryote, intronless genes are
the original form and that introns arose later by
some unknown mechanism.
Organisms which gained introns so that domain
shuffling could occur would be advantaged in the
long term.
What is the origin of split genes?
(2)
•
•
•
A second view is that primitive genes had external,
non-coding regions.
Fusion of genes resulted in intervening regions
which were non-coding.
The evolutionary pressure for rapid division then
resulted in loss of introns in prokaryotes.
Alternative splicing of two (or more)
proteins for the price of one gene
•
A number of cases are known where different
splicing patterns occur with the same initial
transcript. These then lead to different mRNAs
and different proteins.
PolyA tail
A general overview of the differences in the
control of initiation and control of gene
transcription in prokaryotes and eukaryotes
• Unlike in prokaryotes RNA polymerase does not
recognize sites on the DNA itself but binds
because a large number of other proteins bind
and recruit the polymerase.
• A bacterium has about 4000 genes but a mammal
about 30,000, with extensive differentiation into
defined tissues. Nevertheless in nearly all cell
types the DNA is the same.
• Some genes are present in nearly all cells – these
are housekeeping genes.
Types of eukaryotic genes and
their controlling regions
• In eukaryotes there are three different RNA
polymerases, I, II and III.
• Type II transcribes nuclear protein coding genes.
Type I codes for rRNA and type III for tRNA
molecules.
Type II eukaryotic gene
promoters
• Fig 24.16 shows the components of a type II
promoter.
• The basal elements include a short sequence of
pyrimidines (initiator) and the TATA box, which is
at about –25 base pairs.
• Upstream control elements occur between –50
and –200 base pairs. These include the CAAT
box, and the GC box. All genes require at least
one of these but there is no standard pattern.
See Fig. 24.17 for typical examples.
Most transcription factors are
regulated (1)
• Many factors can exist in active and inactive
states. Often activation is via phosphorylation or
dephosphorylation. (See Figs. 24.18 a, b and c
for examples).
• Activation can also be associated with the
movement of the transcription factor to the
nucleus.
• Factors have both DNA binding domains and a
binding domains for other proteins (Fig. 23.8).
Most transcription factors are
regulated (2)
• Commonly protein kinase A activates a
transcription factor by phosphorylation. This
occurs in response to a rise in cAMP.
Regulation
chromatin