Transcript Prok transcription

Transcription
Transcription- synthesis of RNA from only one strand of a double
stranded DNA helix
DNARNA(Protein)
Why is RNA an intermediate????
1. Protect the DNA; limited access;
2. Gives regulatory opportunity (all cells have the same DNA but not
the same genes are expressed)
3. In Eukaryotes the DNA is located in the nucleus and RNA
transports the information out to the protein synthesis apparatus in
the cytoplasm
RNA transcription
•  same general mechanism in prokaryotes and
eukaryotes
•  Differences occur in gene structure
Prokaryotes
 Genes usually code for more than one polypeptide (euks
only one)
 Introns almost non existent in prokaryotes consequently
less mRNA processing in bacteria
 Transcription and translation occur simultaneously in
prokaryotes
 all takes place in cytoplasm in prokaryotes
RNA synthesis is a template dependent process
 The DNA dependent RNA polymerases adds
ribonucleotide units to the 3' end of the growing RNA
chain using one strand of the DNA duplex as a template
 the added ribonucleotides adhere to the base pairing
rules except for the addition of U instead of T
 the RNA has a sequence identical to the non template
strand of DNA except for substitutions of U for T
 Polymerization occurs only in the 5' to 3' direction, as
does DNA synthesis
 No primer is required for transcription
The bacterial RNA polymerase
 consists of a core enzyme containing several
polypeptides
 α, β, and β’ subunits make up the core. This core
enzyme is potentially capable of copying DNA from any
source to RNA
 In order to initiate transcription with high efficiency on
bacterial promoters, the enzyme must combine with
another polypeptide unit, - a sigma factor
 the sigma factor increases the selectivity of the bacterial
RNA polymerase for bacterial promoters by ~ a million
times
 Distinct sigma factors enable RNA polymerases to
distinguish between different bacterial gene groups transcription regulation
RNA synthesis is initiated at specific DNA sequences called promoters
 Promoters are part of bacterial transcription units called operons
 Operons usually contain more than one coding sequence. An
individual coding sequence in an operon is called a cistron
 Most bacterial operons are controlled by a single promoter lying in
the region in front of the transcribed fragment
 Some have 2 or more promoters arranged tandemly in the 5'
flanking region
 different promoters are controlled by different regulatory factors
Consensus sequences
 Bacterial promoters contain consensus sequences that provide
recognition and binding sites for RNA polymerase, regulate the rate
of initiation of transcription and indicate the start point
 2 consensus sequences occur in E coli promoters that are
recognised by RNA polymerase in combination with sigma factor
σ70
 the short sequence TATATT, also called the -10 sequence or Pribnow
box, is usually centered about 10 nucleotides upstream of the start
point.
 TTGACA, called the -35 sequence, usually begins about 35
nucleotides upstream from the start point
Bacterial promoters
 Vary in the strength with which they bind RNA polymerase
 depends on how closely their -10 and -35 sequences fit the
consensus sequence
 those with perfect copies of the consensus sequence bind strongly
 those with base substitutions altering the consensus sequence bind
RNA polymerase more weakly
 The degree of binding of RNA polymerase by the promoter sets
the base level for the initiation of transcription
 This base level is adjusted by 2 types of regulatory proteins called
repressors and activators
Prokaryotic transcription proceeds through initiation,
elongation and termination
1. Initiation
 the core enzyme in combination with a sigma factor
binds strongly to a promoter
 strong binding is quickly followed by the DNA unwinding
and the initiation of transcription at the start point
 The sigma factor is released after transcription begins
2. Elongation
 As elongation begins, NusA (a protein) is loaded on to
the RNAP(RNA polym) complex and sigma factor is
displaced from the complex.
 the core enzyme continues transcribing the operon until
it reaches termination signals at the 3' end of the gene
3. Termination
there are 2 types of sequence signalling termination
1. rho independent termination/intrinsic termination
there is formation of a stable GC rich stem-loop in the newly
synthesized RNA followed by a string of U's (A's in the template
strand) spaced about 20 bases downstream (these sites are often
called intrinsic terminators).
The stem loop "snares" the polymerase, slowing or stalling it.
This pause, coupled with the low stability of the RNA-DNA hybrid at the
active site (run of A=U basepairs) allows the RNA polymerase to
fall off the template DNA and terminates the RNA transcription for
that gene.
2. rho dependent termination
rho binds the newly formed RNA and stalls the RNA polymerase by
interacting with it.
Rho termination activity is stimulated by ATP hydrolysis.
Nus factors (proteins) can interact to enhance termination
1. rho independent termination/intrinsic termination
rho dependent termination
A sequence near the end of
the newly synthesized strand
binds to the rho protein. This
protein physically forces the
release of the RNA.
The Types or Classes of RNA
·
mRNA
o
this is the coded information for how to make a particular protein
§
codon = 3 nucleotide code specifying an amino acid
o
monocistronic – codes for just one protein
o
polycistronic – codes for several proteins
o
mRNA code also has start and stop signals for translation
o
may have an upstream leader sequence that also regulates activity
o
unstable molecule
· rRNA
o
o
o
o
· tRNA
o
o
o
o
o
stable
helps to make up the ribosomes
bacteria have 16s, 23s, and 5s rRNA
undergoes posttranscriptional modification (RNA processing):
§
pieces are cut from a larger primary transcript
stable
carries amino acids to the ribosome for protein synthesis
has an anti-codon that base pairs with the mRNA codon
must have a different tRNA for each amino acid
RNA processing (see fig 8-9):
§
Cut out tRNA from a much longer primary transcript
§
Chemical modification of some bases