Download Transcription. (Ms. Shivani Bhagwat)

Transcription of DNA
Ms. Shivani Bhagwat
Lecturer,
School of Biotechnology
DAVV
Transcription:
information transfer from DNA to RNA
The information encoded by DNA is transcribed into RNA
information of RNA is translated into proteins
Transcription (RNS synthesis) is carried out by RNA polymerases
RNA polymerases are large complexes of proteins, not single enzymes
Transcription is more complex in eukaryotes than in prokaryotes, but
basic mechanisms are very similar in all living cells
Protein coding sequences constitute only 1-2 % of the human genome!
DNA encodes a number of RNA molecules, which are not mRNAs:
ribosomal RNAs, rRNAs
transferRNAs, tRNAs
small nuclear RNAs, snRNAs
small nucleolar RNA, snoRNA
microRNAs, miRNAs
anti-sense RNAs
RNA molecules, that are parts of enzymes
Information transfer from DNA to RNA
Transcription and
translation are closely
linked processes.
In prokaryotes they
are physically linked,
in eukaryotes they
mutually regulate
each other, sharing
controling molecules
and mechanisms.
Cells contain 20 times
more RNA than DNA
Transcription, RNA polymerases
DNA is transcribed by RNA polymerase, a large protein complex, which
synthesizes an RNA strand, complementary to the template strand of DNA.
RNA polymerase has to unwind the DNA over a short distance then moves
stepwise along the
template strand of
DNA.
The newly formed RNA
is only basepaired with
a short stretch of
deoxynucleotides.
RNA polymerase enzymes
are very large:
100 kDa in T7 bacteriophage.
400 kDa in bacteria.
500 kDa for eukaryotes
RNA polymerases
RNA polymerase adds nucleotides one by one to the RNA chain at the
polymerisation site. The polymerase rewinds the two DNA strands behind this site to
displace the newly formed RNA.
A short region of DNA/RNA helix
is therefore formed only transiently
and the RNA transcript is a singlestranded complementary
copy of one of the two
DNA strands.
The incoming nucleotides
are in the form of ribonucleoside triphosphates
(ATP, UTP, CTP, and
GTP), whose hydrolysis
provides the energy for the
polymerisation reaction.
www.csu.edu.au
RNA polymerases
Plastid and mitochondrial RNA polymerases and bacteriophage T7's
RNA polymerase are single-subunit DNA polymerases
All multi-subunit RNA polymerases
have 5 core subunits. The (true)
Bacteria have an additional
σ-(sigma) factor subunit that aids
regulation and binding to DNA.
Eukaryotic RNA polymerases have
five core subunits plus five common
subunits.
RNA polymerases
The prokaryotic holoenzyme has 2 alpha, 2 beta and an omega
subunits. It binds DNA with no sequence specificity.
To find the promoter sequence it needs
another subunit: the sigma factor
Formation of the open complex
from closed DNA is usually the
slowest step in transcription
initiation.
About 10 nucleotides are
polymerized in the open complex
before it must undergo promoter
clearance.
A conformational change must
take place that releases the
sigma factor.
Prokaryotic promoter sequences
Promoter
-35 Region spacer
trp operon TTGACA
N17
tRNA tyr
TTACA
N16
lP2
TTGACA
N17
lac operon TTTACA
N17
rec A
TTGATA
N16
lex A
TTCCAA
N17
T7A3
TTGACA
N17
consensus
TTGACA
TGAC box (-35)
-10 Region start
TTAACT
N7A
TATGAT
N7A
GATACT
N6G
TATGTT
N6A
TATAAT
N7A
TATACT
N6A
TACGAT
N7A
TATAAT
TATAAT Box (-10), This sequence was initially
called the Pribnow Box. Do not refer this
sequence as a TATA box.
All these promoter sequences are recognized by the sigma70 subunit in E. coli.
Note the degree of sequence variation at each position. The consensus sequence has
been derived from a much larger database of over 300 well-characterized promoters.
The "consensus sequence" is a hypothetical sequence made up of the nucleotides
found most often in each position. There may be no single organism with this exact
set of nucleotides in the stated positions.
The promoter sequences are asymmetrical, the promoter will bind the polymerase in
only one orientation, thus determining which strand of the DNA will be transcribed.
RNA synthesis by RNA pol
RNA polymerase synthesizes ribosomal RNAs. In bacteria there are many
(in E. coli 7) ribosomal rRNA genes.
RNA polymerase – as all other NA synthesizing enzymes – polymerizes
from 5’>3’, DNA is read from 3’>5’
The produced RNA is complementary to template strand,
and equivalent to non-template strand
RNA polymerase stops at
termination or stop signal.
Rho factor binds RNA and
helps recognizing
termination signals (RNA loops).
Palindromic sequences (e.g.
AATCG.CGATT) form loops
on the RNA and serve as
signal termination.
The 16S rRNA
The 16S rRNA
The 16S rRNA is 1.5 kb long
and has an elaborate
secondary structure
rRNA molecules in the ribosome
Ribosomes are very
large RNA-protein
complexes, subcellular
organelles.
They are responsible
for the synthesis of
proteins.
Both prokaryotic and
eukaryotic ribosomes
consist of two subunits
and have 3 (4) RNA
molecules.
Processing of the rRNAs
Endonucleolytic cleavage of ribosomal RNA precursors in E. coli. The primary transcript
contains a copy of each of the three ribosomal RNAs and may also contain several tRNA
precursors.
The large rRNA precursors are cleaved from the large primary transcript by the action of RNase III.
The ends of the 16S, 23S, and 5S rRNAs are trimmed by the action of endonucleases M16, M23,
and M5, respectively.
(Slash marks indicate that portions of the rRNA primary transcript have been deleted for clarity.)
Processing of tRNA molecules
tRNAs and other small RNA molecules are produced by processing
and modification of larger precursor molecules. This is the processing
of a bacterial tRNA.
Synthesis of prokaryotic mRNAs
Genes coding for proteins are first transcribed into mRNAs. One gene
codes for a protein. In cases of multi-subunit proteins with heterologous subunits, one cistron codes for a polypeptide chain. (A protein
with four different subunits is coded by four cistrons.)
In prokaryotes there are single cistronic and polycistronic messages,
coding for one protein or a chain of (related) enzymes. Metabolically
linked enzymes (e.g. enzymes of a synthetic pathway) are usually
controlled together.
Polycistronic mRNAs
are characteristic of
prokaryotic organisms.
Prokaryotic regulation of transcription
There are genes, which are
expressed constitutively.
Others are under control:
synthetic enzymes are negatively
regulated (switched off) if their
product is present, enzymes with
catabolic functions are induced
(swithed on) if their substrate is
available.
Bacteria with continuously
changing environment have more
regulatory units, than those,
which live in more stable niche.
The lac operon
Regulatory elements of the eukaryotic promoter
A TATA Box, located approximately 25 bp upstream of the startpoint of transcription is found
in many promoters. The consenus sequence of this element is TATAAAA, it resembles the
TATAAT sequence of the prokaryotic -10 region (please do not mix them up!)
TATA box appears to be more important for selecting the startpoint of transcription (i.e.
positioning the enzyme) than for defining the promoter.The Initiator is a sequence that is
found in many promoters and defines the startpoint of transcription.
The GC box is a common element in eukaryotic class II promoters. Its consensus sequence is
GGGCGG. It may be present in one or more copies which can be located between 40 and
100 bp upstream of the startpoint of transcription. The transcription factor Sp1 binds to the
GC box.The CAAT box - consensus sequence CCAAT - is also often found between 40
and 100 bp upstream of the startpoint of transcription. The transcription factor CTF or NF1
binds to the CAAT box.
The eukaryotic RNA pol I
In eukaryotes RNA polymerase I transcribes rRNA genes. The CORE promoter region is
located
from -31 to +6 around the
transcription startpoint.
Other sequences further
upstream, called the upstream control elements
(UCE) in the promoterproximal region located
from -187 to -107 are
also required for efficient
transcription.
The eukaryotic RNA pol I
Assembly of a eukaryotic transcriptional
complex. Two additional transcription factors are
known to be required to assist RNA polymerase I.
UBF1 is a single polypeptide which binds to the
upstream control element (UCE) and to the CORE
promoter. UBF1 recognizes a GC-rich sequence
within these elements. UBF1 is an assembly
factor.
SL1 binds to UBF1. It consists of 4 proteins, one of
which is TATA-box binding protein (TBP). TBP is
required for the assembly of a transcriptional
complex in all 3 classes of eukaryotic transcription
units. SL1 is a positional factor - it targets RNA
polymerase at the promoter so that it initiates
transcription in the correct place. Once UBF1 and
SL1 have formed a complex, RNAP I binds to the
CORE promoter to initiate transcription
RNA polymerase II
RNA polymerase II All genes that are transcribed and expressed via mRNA are transcribed
by RNA polymerase II. Transcription copies the DNA code of a gene and converts it to
high mol mass nuclear RNA (hnRNA), which is precessed to mRNA. The mRNA will be
used at the ribosome to make polypeptides (proteins).
RNA polymerase II is a multisubunit enzyme-complex.The yeast enzyme has 12 subunits.
The largest subunit is the homologue of the beta subunit of the prokaryotic enzyme, it
contains the catalytic activity. The enzyme is very sensitive to amanitin, a mushroom toxin.
RNA polymerase II can transcribe RNA from nicked dsDNA templates or from ssDNA
templates. However, it cannot initiate transcription at a promoter. In this respect, it
resembles the core form of bacterial RNA polymerase.
The promoters used by RNA polymerase II have different structures depending upon the
particular combination of transcription factors that are required to build a functional
transcriptional complex at each promoter. Nevertheless, these different structures can be
viewed as a combination of a relatively limited number of specific sequence elements.
RNA pol II
The transcription complex. The fact
that the enzyme can not initiate
transcription correctly on a dsDNA
template indicates that RNA
polymerase II needs additional
transcription factors.
At least six general transcription factors
(TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH) have been characterized. In the
presence of these transcription factors,
the enzyme is able to initiate
transcription at promoters correctly.
However, even in the presence of
transcription factors, the enzyme
complex is unable to recognize and
respond to regulatory signals.
The eukaryotic RNA pol II
rotation of DNA:
www.k2.phys.waseda.ac.jp/Rnapmovies/RNAP.htm
The RNA pol II transcription complex
In addition to the general transcription factors, the eukaryotic transcriptional complex will also be
affected by the presence of an promoter-proximal regulatory sequences (transcription elements
or motifs) and the presence of transcription factors that bind to those sequences.
Such factors may be
present in some
cells/tissues but
not in others.
.
hnRNA and mRNA
All of the code contained in the hnRNA molecule is not needed to produce
the polypeptide. The sections of hnRNA which do not code for translation of
polypeptide are called introns and untranslated region.
As the mRNA readies itself to leave the nucleus, enzymes cut out and
remove the introns. The remaining exons are spliced back together again
by a different enzyme. This modified mRNA is what comes to the ribosome
to be translated into polypeptides.
Eukaryotic transcription and mRNA synthesis is not taking place in discrete
steps: transcription, capping, tailing, splicing and export from the nucleus
for translation. The contemporary view of eukaryotic gene expression
entails simultaneous transcription and processing..
Molecular mechanisms of splicing
Splicing, splicesomes
RNA-protein complexes
(splicesomes) catalyse
the removal of introns
from the hnRNAs.
There are consensus
sequences for donor and
acceptor sites (5’ and 3’
ends of exons) and also
for a branch site.
Splicing, splicesomes
mRNAs usually contain many introns
Only eukaryotic mRNAs do contain
exons and introns. The are single
intron mRNAs, but most proteins
are coded by multi-intron mRNAs.
Certain introns (or homologues) can
be found in many proteins of
similar functions.
In different tissues (or in
developmental stages) different
splice variants of the same mRNA
can be found (alternative splicing).
Alternative splicing
Alternative splicing makes the genome
more flexible and economical: with one
gene many proteins of different functions
can be coded. One example is sex
determination in Drosophila.
Capping and polyadenylating
mRNA molecules undergo other types of modifications.
To increase stability, a „cap” structure
(a mG of reverse orientation) is added
to the 5’ end: this way the molecule
has only 3’ ends!
The 3’ polyadenylation sequence is bound
by a cleavage and polyadenylation
specificity factor (CPSF). An endonuclease
binds to CPSF and cleaves the transcript.
Finally a polymerase binds to CPSF,
adding up to 250 adenylate residues to the
3' end of the transcript. (This polymerase
does not need a template!) The length of the
polyA tail regulates translational activity of the mRNA.
Some viral mRNAs do not contain caps: instead, a protein is linked to the mRNA
(covalently).
RNA polymerase III
RNA polymerase transcribes
tRNAs and other low MM RNAs,
usually in a precursor form.
tRNA molecules are adaptors
each amino acid has at least one
tRNA, which brings aa’s to the
ribosomes.
Aminoacyl-tRNA synthetases
charge specific tRNAs with
cognate amino acids
More tRNAs serve one amino
acid
Processing of the tRNAs
The transcription product, the pre-tRNA, contains additional RNA sequences at both the 5’ and 3’-ends. The
additional nucleotides at the 5’-end are removed by an unusual RNA containing enzyme called
ribonuclease P (RNase P).
An intron is removed from the tRNA mol.
All mature tRNAs contain the trinucleotide
CCA at their 3’-end. These bases are not
coded for by the tRNA gene. Instead,
these nucleotides are added to the
pre-tRNA transcript. The enzyme
responsible for the addition of the
CCA-end is tRNA nucleotidyl transferase
Mature tRNAs can contain up to 10% bases
other than the usual A, G, C and U.
These base modifications are introduced
into the tRNA at the final processing step.
tRNA molecules
Models of tRNA. The tRNAs contain
special bases (not found in other
NAs), these are modified after
synthesis. The bases denoted psi
(pseudouridine) and D (dihydrouridine) are derived from uracil.
Functional domains of the tRNA molecule.
The acceptor stem is aminoacylated, the
anticodon arm recognizes the codon of
the mRNA.
The figure shows how a tRNA molecule is
shaped due to basepair interactions. Note
the specificity due to the anticodon and
due to the fact that each tRNA is charged
with a specific amino acid.
RNA polymerase III
transcribes genes encoding
small structural RNAs that
include 5S RNA, tRNA and the
U6 and 7SK RNAs.
Together with RNA
polymerase III, transcription
factor IIIC (TFIIIC) and TFIIIB
are sufficient for the
transcription of tRNA, and
yeast U6 RNA genes.
Nucleosome remodeling
factors are complex
molecular machines, which
modulate histone/DNA
interactions in order to
enhance the accessibility of
nucleosomal DNA.
MicroRNAs, antisense and gene silencing
The discovery of microRNAs and the
phenomenon of RNA interference
might change our whole idea of gene
expression and gene regulation.
The human genome codes for a large
number of microRNA sequences, their
importance is just emerging.
It was known that only a small fraction of
mRNAs leave the nucleus and
participate in protein synthesis.