Transcript m5zn_a4ac3a22336dedd

Transcription in Prokaryotic
(Bacteria)
• The conversion of DNA into an RNA transcript
requires an enzyme known as RNA
polymerase
• RNA polymerase
– Catalyzes the formation of a phosphodiester bond
between the 3’ end of the growing mRNA chain
and the new ribonucleotides
– Transcription must occur in the 5’ to 3’ direction
– No primer is needed to begin transcription
Transcription in Bacteria
The template strand on DNA is what is “read” by RNA polymerase into a message.
The non-template strand is confusingly referred to also as the coding strand because
the DNA sequence in this strand is complementary to the message which is made into
Protein.
RNA Polymerase Structure
• X-ray crystallography
indicates that
– Large globular enzyme
– Several channels in the
interior
• Core enzyme
– Just RNA polymerase
– Active site for catalysis
• Holoenzyme
– Sigma plus RNA
polymerase
Holoenzyme
Initiation of Transcription in Bacteria
• Promoters
– Sequences of DNA which “inform” RNA
polymerase where to begin transcription
– Located on non-template strand
– 40 to 50 base pairs in length
– All have a TATAAT sequence
• -10 box centered 10 bases away from the site where
transcription begins (+1)
– All have a TTGACA
• -35 box located 35 bases away from the +1 site
– Sequences inside promoter but outside -10 or -35
vary widely among genes
Terms
• Downstream
– DNA located in the direction in which RNA
polymerase will transcribe
• Upstream
– DNA located in the opposite direction in which
RNA polymerase will transcribe
Upstream
Downstream
Promoter
TTGAC
-35
TATAAT
-10
Gene
+1
Initiation of Transcription in Bacteria
• Sigma makes the initial contact with DNA that
starts transcription in bacteria
• Once sigma has bound to the DNA the helix
opens and the template is threaded through a
channel
• NTPs enter a different channel and diffuse to
the active site
• Incoming NTPs base pair complementary with
the template strand
– A base pairs with U not T
Opens the helix
Steers the template and
non-template strands into
the correct channels
Termination of Transcription in
Bacteria
• DNA sequence functions as a transcription
termination signal
• Makes a stretch of RNA which as soon as it is
synthesized can base pair with itself
– Hairpin loop – RNA secondary structure
• The formation of the hairpin disrupts the
interaction between RNA polymerase and the
RNA transcript resulting in the physical
separation
Termination of Transcription in
Bacteria
Transcription in Eukaryotic Cells
• Transcription of eukaryotic cells is more
complex and requires more proteins and
regulators
• Transcription factors
• The initiation of transcription is accomplished
by basal transcription factors
– Function analogous to sigma
– Can interact with DNA independently of RNA
polymerase
Transcription Regulation
• What we know from prokaryotes:
– Several related genes can be transcribed together (ie. lac
operon)
– Need RNA Polymerase to recognize a promoter region
• Why eukaryotes are different:
– Genes are nearly always transcribed individually
– 3 RNA Polymerases occur, requiring multiple proteins to
initiate transcription
Transcription Regulation
• Typical prokaryotic promoter: recognition
sequence + TATA box -> RNA Polymerase
attachment -> transcription
• Typical eukaryotic promoter: recognition
sequence + TATA box + transcription factors ->
RNA Polymerase II attachment -> transcription
Transcription Regulation
• RNA polymerase interacts w/promoter, regulator
sequences, & enhancer sequences to begin
transcription
– Regulator proteins bind to regulator sequences to activate
transcription
• Found prior to promoter
– Enhancer sequences bind activator proteins
• Typically far from the gene
• Silencer sequences stop transcription if they bind
with repressor proteins
Activator:
A transcriptional activator is a protein that increases gene
transcription of a gene or set of genes.
Most activators are DNA-binding proteins.
Most activators function by binding sequence-specifically to a DNA
site located in or near a promoter and making protein-protein
interactions with the general transcription machinery (RNA
polymerase and general transcription factors).
Transcription factor :
is a protein that binds to specific DNA sequences, thereby controlling
the flow (or transcription) of genetic information from DNA to mRNA.
Transcription factors perform this function alone or with other
proteins in a complex, by promoting (as an activator), or blocking (as
a repressor) the recruitment of RNA polymerase (the enzyme that
performs the transcription of genetic information from DNA to RNA)
to specific genes
Repressor:
repressor is a DNA-binding protein that regulates the expression of
one or more genes by binding to the operator and blocking the
attachment of RNA polymerase to the promoter, thus preventing
transcription of the genes. This blocking of expression is called
repression.
Repressor proteins are coded for by regulator genes. Repressor
proteins then attach to a DNA segment known as the operator. By
binding to the operator, the repressor protein prevents the RNA
polymerase from creating messenger RNA
Transcription in Eukaryotic Cells
• Eukaryotic genes contain promoters
• Promoters are much more diverse and
complex
• Many promoters recognized by RNA pol II
include a TATA box at -30
• Some promoters recognized by RNA pol II do
not contain a TATA box
• RNA pol I and RNA pol III interact with entirely
different promoters
Transcription in Eukaryotic Cells
• Unlike prokaryotic cells eukaryotic cells must
process the mRNA in the nucleus before it can
be made into a protein
• Three major modifications are made
– Intron splicing
– Addition of poly A tail
– Additon of 5’ cap
Exons & Introns
• Exons
– Coding regions of DNA
– Expressed as proteins
• Introns
– Non-coding regions of DNA
– Intervening
Intron Removal
• Introns are removed in a process known as intron
splicing
• Splicing occurs while transcription is still underway
• Removed by ribozymes known as small nuclear
ribonucleoproteins
– snRNPs :
are RNA-protein complexes that combine with unmodified
pre-mRNA and various other proteins to form a
spliceosome, a large RNA-protein molecular
complex upon which splicing of pre-mRNA occurs.
The action of snRNPs is essential to the removal of
introns from pre-mRNA, a critical aspect of posttranscriptional modification of RNA, occurring only
in the nucleus of eukaryotic cells.
1- Pre-mRNA splicing.
Small nuclear RNAs (snRNAs)
form a complex called a
spliceosome with small nuclear
ribonucleoproteins (snRNPs)
and other proteins.
2- The snRNAs bind to specific
nucleotides in the introns of a
pre-mRNA.
3- The RNA transcript is cut,
releasing the introns and
splicing the exons together,
producing mature mRNA.
Adding a Cap and a Tail
• 5’ cap
The process of 5′ capping is vital to creating mature messenger
RNA, which is then able to undergo translation. Capping ensures
the messenger RNA's stability while it undergoes translation in the
process of protein synthesis, and is a highly regulated process that
occurs in the cell nucleus. Because this only occurs in the nucleus,
mitochondrial and chloroplast mRNA are not capped.
• Poly A tail
– Polyadenylation is the addition of a poly(A) tail to an RNA
molecule. The poly(A) tail consists of multiple adenine bases. In
eukaryotes, polyadenylation is part of the process that produces
mature messenger RNA (mRNA) for translation. It, therefore,
forms part of the larger process of gene expression.
Comparing Transcription: Bacteria vs. Eukaryotes
Aspect
RNA polymerase
Bacteria
One
Eukaryotes
Three; each produces a
different class of RNA
Promoter
Structure
Complex and variable;
-35; -10
often has TATA @ -30
Proteins which
contact promoter
Sigma;
Many basal transcription
many
factors
types
RNA processing
None
Extensive
1. Intron splicing
2. Cap and poly A tail