Transcript Transcription - Bilkent University

Transcription
Central Dogma
from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates
Types of RNA
• messenger RNA (mRNA). This will later be translated
into a polypeptide.
• ribosomal RNA (rRNA). This will be used in the
building of ribosomes
• transfer RNA (tRNA). RNA molecules that carry amino
acids to the growing polypeptide.
• small nuclear RNA (snRNA). DNA transcription of the
genes for mRNA, rRNA, and tRNA produces large
precursor molecules ("primary transcripts") that must
be processed within the nucleus to produce the
functional molecules for export to the cytosol. Some of
these processing steps are mediated by snRNAs.
• small nucleolar RNA (snoRNA). These RNAs help
process ribosomal RNA (rRNA) molecules.
Ribosomal RNA (rRNA)
• There are 4 kinds. In
eukaryotes, these are
• 18S rRNA. One of these
molecules, along with
some 30 different
protein molecules, is
used to make the small
subunit of the ribosome.
• 28S, 5.8S, and 5S rRNA.
One each of these
molecules, along with
some 45 different
proteins, are used to
make the large subunit
of the ribosome.
Transfer RNA (tRNA)
• There are 32 different kinds of
tRNA in a typical eukaryotic
cell.
• each is the product of a separate
gene
• many of the bases in the chain
pair with each other forming
sections of double helix. the
unpaired regions form 3 loops
• each kind of tRNA carries (at its
3' end) one of the 20 amino
acids (thus most amino acids
have more than one tRNA
responsible for them) at one
loop, 3 unpaired bases form an
anticodon
Messenger RNA (mRNA)
• Messenger RNA comes in a wide range of sizes
reflecting the size of the polypeptide it encodes.
Most cells produce small amounts of thousands
of different mRNA molecules, each to be
translated into a peptide needed by the cell.
• Many mRNAs are common to most cells,
encoding "housekeeping" proteins needed by
all cells (e.g. the enzymes of glycolysis). Other
mRNAs are specific for only certain types of
cells. These encode proteins needed for the
function of that particular cell (e.g., the mRNA
for hemoglobin in the precursors of red blood
cells).
Transcription
from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates
Transcription in Prokaryotes
• Requires a promoter region, with which
RNA polymerase interact.
• The RNA-coding sequence is the DNA that
is to be transcribed and later translated.
• Requires a terminator, downstream of the
end of the RNA-coding sequence.
Promoter
• Specific sequences are critical for the recognition
by the polymerase.
• Generally found at –35 and –10; that is they are
centered at 35 and 10 bp upstream of +1,
transcription start site.
• Consensus sequence for –35 region (-35 box) is
5’-TTGACA-3’
• Consensus sequence for –10 region (Pribnow box)
is 5’-TATAAT-3’.
RNA Polymerase
• A form of RNA polymerase called holoenzyme
(Complete enzyme) must bind to the promoter.
• Holoenzyme consists of the the core enzyme
(made up of four polypeptides) bound with
another polypeptide called the sigma factor,
necessary to recognize the –35 and –10 regions of
the promoter.
• Without the sigma factor, the core enzyme doesn’t
start transcription efficiently.
Binding to the promoter
• Binds in two steps
– Binds loosely to the –35 box while DNA is
double stranded.
– Binds more tightly to DNA as DNA untwists
for about 17 bp centered around the –10 box.
Promoter sequences and
regulation of transcriptional rate
• Promoters differ in their sequences slightly,
so the binding efficiency of the promoter
varies.
• A –10 region sequence of 5’-GATACT-3’
has a lower rate of transcription initiation
than 5’-TATAAT-3’.
Sigma factor and transcriptional
regulation
• There are different sigma factors; sigma 70
is the most common
• sigma32 is used for conditions of heat
shock; sequences that are recognized by
sigma32 has CCCCC at –39 and
TATAAATA at –15.
Elongation
• Transcription bubble forms.
• Once 8 or 9 RNA nucleotides are linked together,
the sigma factor disassociates from the RNA
polymerase core enzyme.
• Core enzyme completes the transcription. As it
moves, it untwists the DNA double helix
producing torsion, DNA double helix reforms
behind the enzyme.
• Within the untwisted region a temporary RNADNA hybrid is formed, the rest of the RNA is
displaced away from the DNA.
Termination
• Requires terminator sequences.
– Rho-independent termination. These sequences consists
of sequences with a two-fold symmetry that makes a
hairpin loop, followed by 4-8 AT basepairs, leading to
termination when encountered.
– Rho-dependent termination. These sequences lack AT
string and many cannot form hairpin structures. Rho
factor (a protein) is needed to recognize a specific
sequence and face the polymerase by moving along the
DNA using ATP.
Transcription in Prokaryotes
Enzyme: RNA polymerase
Promoter is the DNA sequence where RNA polymerase
binds to initiate transcription
Transcription in E.Coli
The Operon Model
• Groups of genes
coding for related
proteins are
arranged in units
known as operons.
• An operon consists of
an operator,
promoter, regulator,
and structural genes.
The Operon Model
• The regulator gene codes
for a repressor protein
that binds to the
operator, obstructing the
promoter (thus,
transcription) of the
structural genes.
The regulator does not
have to be adjacent to
other genes in the
operon. If the repressor
protein is removed,
transcription may occur.
Eukaryotic Transcription
RNA Processing:
pre-mRNA -> mRNA
• Synthesis of the cap. This is a stretch of
three modified nucleotides
(7methylguanosine) attached to the 5' end
of the pre-mRNA. This structure aligns
mRNAs on the ribosome during
translation
Capping
RNA Processing:
pre-mRNA -> mRNA
• Synthesis of the poly(A) tail. This is a
stretch of adenine nucleotides attached to
the 3' end of the pre-mRNA.
• This regulates both translation and
mRNA stability. It is also important in
early development.
Polyadenylation
RNA Processing:
pre-mRNA -> mRNA
• Splicing. Step-by-step removal of introns
present in the pre-mRNA and splicing of
the remaining exons.
Splicing
Splicing
• Three critical sequence elements of premRNA:
• Sequences at the 5’ splice site
• sequences at the 3’ splice site
• sequences within the intron at the branch point
Spliceosomes
• Composed of proteins and RNAs.
• The RNA components of the spliceosome
are small nuclear RNAs (snRNAs) called,
U1, U2, U4, U5, and U6. These range
between 50 to 200 nucleotides complexed
with six to ten protein molecules to form
small nuclear ribonucleoprotein particles
(snRNPs).
Self-splicing
• Some RNAs are capable of self-splicing.
• They can catalyze the removal of their own
introns in the absence of other RNA or
proteins.
• 28S RNA of the protozoan Tetrahymena.
• Self-splicing is catalyzed by the intron
itself, which acts as a ribozyme.
Alternative splicing
The RNA polymerases
• RNA polymerase I (Pol I). Located in
nucleolus. It transcribes the rRNA genes for
the precursor of the 28S, 18S, and 5.8S
molecules (and is the busiest of the RNA
polymerases). S values are derived from the
rate at which the rRNA molecules sediment
during sucrose gradient centrifugation.
The RNA polymerases
• RNA polymerase II (Pol II). Found in
nucleoplasm. It transcribes the mRNA and
snRNA genes.
• RNA polymerase III (Pol III). Found in
nucleoplasm. It transcribes the 5S rRNA
genes and all the tRNA genes.
mRNA transcription by RNA
PolII
• RNA polyII produces a precursor mRNA (premRNA).
• Promoters contain basal promoter elements and
promoter proximal elements, having general activity.
– Basal promoter elements: TATA box is located at about
position –25, and a pyrimidine-rich sequence near the
transcription start site is called the initiator element (Inr).
TATA box consensus is TATAAAA.
– Promoter proximal elements: Located upstream of TATA
box, about 50-200 nucleotides from the start of
transcription. Examples are CAAT (‘cat’) box, which is
located at about –75; GC box with a consensus of
GGGCGG, located at about –90.
Transcription Factors
• Basal transcriptional factors are the
proteins that are required for the function
of RNA polymerase II
• They bind to the promoter area, TATA Box
• General Transcription Factors
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TFIID
TFIIB
TFIIF
TFIIF
TFIIH
Transcription Factors
• TFIID binds to the TATA box to form the initial
committed complex. TFIID has a subunit called TATAbinding protein (TBP) that recognizes the TATA box,
and a number of other proteins called TBP-associated
factors (TAFs).
• TFIID-TATA box complex acts as a binding site for
TFIIB, which recruits RNA PolII and TFIIF to produce
the minimal transcription initation complex.
• Next, TFIIE and TFIIH bind to produce the complete
transcription initiation complex (or preinitiation
complex, PIC).
Other transcription factors
• Activators bind to enhancers, sequences required
for maximal transcription.
• Activator binds to an enhancer, and through an
interaction with another protein called an adapter
forms a bridge to the preinitation complex.
• In most cases, enhancers are upstream of the gene.
• Silencer elements have repressing ability for the
transcription upon binding with a repressor.
Transcription by RNA Pol I
• Transcription of rRNA genes.
• rRNA genes are found in tandem repeats.
• A large 45S pre-RNA is transcribed, then processed to
yield 28S, 18S, and 5.8S rRNAs.
• Promoter spans about 150 bp upstream of the
initiation site.
• UBF (upstream binding factor) and SL1 (selectivity
factor) are transcription factors that recognize the
promoter.
• TBP is a component of SL1 and is essential.
• rDNA promoter does not contain TATA box.
rRNA gene
rDNA transcription
Transcription by RNA Pol III
• Synthesizes tRNA and 5S rRNA, and some small
RNAs involved in splicing and protein transport.
• Promoter of 5S rRNA is downstream of the
transcription initiation site!
– TFIIIA, TFIIIC, TFIIIB, and the polymerase bind to the
promoter, sequentially.
• tRNA promoters do not contain a binding site for
TFIIIA.
– TFIIIC initiates transcription.
• The promoter of U6 snRNA is upstream of the start
site and contains a TATA box, recognized by TBP
subunit of TFIIIB.
Transcription of polymerase III
genes
RNA processing
RNA processing
• Three of the Eukaryotic rRNAs (28S, 18S,
and 5.8S rRNAs) are cleaved from a single
transcript.
• Bacterial rRNAs (23S, 16S, and 5S) are
cleaved from a single transcript.
• Cleavage involves several steps are
somewhat different in eukaryotes and
prokaryotes.
Processing of rRNAs
Processing of tRNAs
• tRNAs are synthesized as longer molecules,
pre-tRNAs, some of which contain multiple
tRNAs.
• The 5’ end of pre-tRNA is cleaved by an
enzyme called RNaseP.
• RNaseP contains RNA and proteins, and
RNA itself can do the cleavage.
• RNaseP is a ribozyme.
Processing of tRNAs
• The 3’ of tRNAs is generated by Rnase.
• The CCA is added into the terminus, as the site of
amino acid attachment, and is required for tRNA
activity during translation.
• Some bases (10%) are modified at characteristics
places, e.g., methylguanosine, inosine, etc. They
alter the base pairing properties of the tRNA
molecule.
Processing of tRNA
Processing of tRNA
Nonsense-mediated mRNA decay
• A quality control system
• Degredation of mRNAs that lack complete
open reading frames.
• Takes place in cytoplasm in yeast.
• If a ribosome encounters a premature stop
codon, this system is triggered.
• In mammals, it may take place in nucleus.
RNA degradation in cytoplasm
• rRNA and tRNA are stable; they are found
in high levels in the cell.
• Bacterial mRNA have a half life of 2-3
minutes.
• Why?
• In eukaryotes, different mRNAs have
different life spans (30 min to 20 hours).
How mRNA is degraded?
• Degradation is initiated by shortening of
their polyA tails.
• 5’ cap is removed.
• RNA is degraded by nucleases from both
ends.
• Which RNAs are expected to be short
lived?
RNA degradation
• Shortening of poly A
tails
• Removal of 5’ cap
• degradation of RNA by
nucleases.
• IRE: Iron-response
element.
• If iron is available,
nuclease degrade the
mRNA.
• If iron is scarce, IRE-BP
binds to a sequence near
3’ protect the mRNA.