Title: Transcription
1 2Central Dogma
3Genes
- Sequence of DNA that is transcribed.
- Encode proteins, tRNAs, rRNAs, etc..
- Housekeeping genes encode proteins or RNAs that
are essential for normal cellular activity. - Simplest bacterial genomes contain 500 to 600
genes. - Mulitcellular Eukaryotes contain between 15,000
and 50,000 genes.
4Types of RNAs
- tRNA, rRNA, and mRNA
- rRNA and tRNA very abundant relative to mRNA.
- But mRNA is transcribed at higher rates than rRNA
and tRNA - Abundance is a reflection of the relative
stability of the different forms of RNA
5RNA Content of E. coli Cells
type Steady State Levels Synthetic Capacity Stability
rRNA 83 58 High
tRNA 14 10 High
mRNA 3 32 Very Low
6Phases of Transcription
- Initiation Binding of RNA polymerase to
promoter, unwinding of DNA, formation of primer. - Elongation RNA polymerase catalyzes the
processive elongation of RNA chain, while
unwinding and rewinding DNA strand - Termination termination of transcription and
disassemble of transcription complex.
7E. Coli RNA Polymerase
- RNA polymerase core enzyme is a multimeric
protein a2,b, b, w - The b subunit is involved in DNA binding
- The b subunit contains the polymerase active site
- The a subunit acts as scaffold on which the other
subunits assemble. - Also requires s-factor for initiation forms holo
enzyme complex
Site of DNA binding and RNA polymerization
8s-factor
- The s-factor is required for binding of the RNA
polymerase to the promoter - Association of the RNA polynerase core complex w/
the s-factor forms the holo-RNA polymerase
complex - W/o the s-factor the core complex binds to DNA
non-specifically. - W/ the s-factor, the holo-enzyme binds
specifically with high affinity to the promoter
region - Also decreases the affinity of the RNA polymerase
to non-promoter regions - Different s-factors for specific classes of genes
9General Gene Structure
- Promoter sequences recognized by RNA polymerase
as start site for transcription. - Transcribed region template from which mRNA is
synthesized - Terminator sequences signaling the release of
the RNA polymerase from the gene.
10Gene Promoters
- Site where RNA polymerase binds and initiates
transcription. - Gene that are regulated similarly contain common
DNA sequences (concensus sequences) within their
promoters
11Important Concensus Sequences
- Pribnow Box position 10 from transcriptional
start - -35 region position 35 from transcriptional
start. - Site where s70-factor binds.
12Other s-Factors
- Standard genes s70
- Nitrogen regulated genes s54
- Heat shock regulated genes s32
13How does RNA polymerase finds the promoter?
- RNA polymerase does not disassociate from DNA
strand and reassemble at the promoter (2nd order
reaction to slow) - RNA polymerase holo-enzyme binds to DNA and scans
for promoter sequences (scanning occurs in only
one dimension, 100 times faster than diffusion
limit) - During scanning enzyme is bound non-specifically
to DNA. - Can quickly scan 2000 base pairs
14Transcriptional Initiation
- Rate limiting step of trxn.
- Requires unwinding of DNA and synthesis of
primer. - Conformational change occurs after DNA binding of
RNA polymerase holo-enzyme. - First RNA Polymerase binds to DNA
(closed-complex), then conformational change in
the polymerase (open complex) causes formation of
transcription bubble (strand separation).
15Initiation of Polymerization
- RNA polymerase has two binding sites for NTPs
- Initiation site prefers to binds ATP and GTP
(most RNAs begin with a purine at 5'-end) - Elongation site binds the second incoming NTP
- 3'-OH of first attacks alpha-P of second to form
a new phosphoester bond (eliminating PPi) - When 6-10 unit oligonucleotide has been made,
sigma subunit dissociates, completing
"initiation - NusA protein binds to core complex after
disassociation of s-factor to convert RNA
polymerase to elongation form.
16Transcriptional Initiation
Closed complex
Open complex
Primer formation
Disassociation of s-factor
17Chain Elongation
- Core polymerase - no sigma
- Polymerase is accurate - only about 1 error in
10,000 bases - Even this error rate is OK, since many
transcripts are made from each gene - Elongation rate is 20-50 bases per second -
slower in G/C-rich regions (why??) and faster
elsewhere - Topoisomerases precede and follow polymerase to
relieve supercoiling
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19Transcriptional Termination
- Process by which RNA polymerase complex
disassembles from 3 end of gene. - Two Mechanisms Pausing and rho-mediated
termination
20Pausing induces termination
- RNA polymerase can stall at pause sites
- Pause sites are GC rich (difficult to unwind)
- Can decrease trxn rates by a factor of 10 to 100.
- Hairpin formation in RNA can exaggerate pausing
- Hairpin structures in transcribed RNA can
destabilize DNARNA hybrid in active site - Nus A protein increases pausing when hairpins
form.
3end tends to be AU rich easily to disrupt
during pausing. Leads to disassembly of RNA
polymerase complex
21Rho Dependent Termination
- rho is an ATP-dependent helicase
- it moves along RNA transcript, finds the
"bubble", unwinds it and releases RNA chain
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23Eukaryotic Transcription
- Similar to what occurs in prokaryotes, but
requires more accessory proteins in RNA
polymerase complex. - Multiple RNA polymerases
24Eukaryotic RNA Polymerases
type Location Products
RNA polymerase I Nucleolus rRNA
RNA polymerase II Nucleoplasm mRNA
RNA polymerase III Nucleoplasm rRNA, tRNA, others
Mitochondrial RNA polymerase Mitochondria Mitochondrial gene transcripts
Chloroplast RNA polymerase Chloroplast Chloroplast gene transcripts
25Eukaryotic RNA Polymerases
- RNA polymerase I, II, and III
- All 3 are big, multimeric proteins (500-700 kD)
- All have 2 large subunits with sequences similar
to ? and ?' in E.coli RNA polymerase, so
catalytic site may be conserved
26Eukaryotic Gene Promoters
- Contain AT rich concensus sequence located 19 to
27 bp from transcription start (TATA box) - Site where RNA polymerase II binds
27RNA Polymerase II
- Most interesting because it regulates synthesis
of mRNA - Yeast Pol II consists of 10 different peptides
(RPB1 - RPB10) - RPB1 and RPB2 are homologous to E. coli RNA
polymerase ? and ?' - RPB1 has DNA-binding site RPB2 binds NTP
- RPB1 has C-terminal domain (CTD) or PTSPSYS
- 5 of these 7 have -OH, so this is a hydrophilic
and phosphorylatable site
28More RNA Polymerase II
- CTD is essential and this domain may project away
from the globular portion of the enzyme (up to 50
nm!) - Only RNA Pol II whose CTD is NOT phosphorylated
can initiate transcription - TATA box (TATAAA) is a consensus promoter
- 7 general transcription factors are required
29Transcription Factors
- Polymerase I, II, and III do not bind
specifically to promoters - They must interact with their promoters via
so-called transcription factors - Transcription factors recognize and initiate
transcription at specific promoter sequences
30Transcription Factors
- TFAIIA, TFAIIB components of RNA polymerase II
holo-enzyme complex - TFIID Initiation factor, contains TATA binding
protein (TBP) subunit. TATA box recognition. - TFIIF (RAP30/74) decrease affinity to
non-promoter DNA
31Eukaryotic Transcription
- Once initiation complex assembles process similar
to bacteria (closed complex to open complex
transition, primer formation) - Once elongation phase begins most transcription
factor disassociate from DNA and RNA polymerase
II (but TFIIF may remain bound). - TFIIS Elongation factor binds at elongation
phase. May also play analogous role to NusA
protein in termination.
32- Transcriptional Regulation and
- RNA Processing
33Gene Expression
- Constitutive Genes expressed in all cells
(Housekeeping genes) - Induced Genes whose expression is regulated by
environmental, developmental, or metabolic
signals.
34Regulation of Gene Expression
RNA Processing
mRNA
RNA Degradation
AAAAAA
5CAP
Active enzyme
Post-translational modification
Protein Degradation
35Transcriptional Regulation
- Regulation occurring at the initiation of
transcription. - Involves regulatory sequences present within the
promoter region of a gene (cis-elements) - Involves soluble protein factors (trans-acting
factors) that promote (activators) or inhibit
(repressors) binding of the RNA polymerase to the
promoter
36Cis-elements
- Typically found in 5 untranscribed region of the
gene (promoter region). - Can be specific sites for binding of activators
or repressors. - Position and orientation of cis element relative
to transcriptional start site is usually fixed.
37Enhancers
- Enhancers are a class of cis-elements that can be
located either upstream or downstream of the
promoter region (often a long distance away). - Enhancers can also be present within the
transcribed region of the gene. - Enhancers can be inverted and still function
- 5-ATGCATGC-3 5-CGTACGTA-3
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39Two Classes of Trans-Acting Factors
- Activators and repressors- Bind to cis-elements.
- Co-activators and co-repressors bind to
proteins associated with cis-elements. Promote or
inhibit assembly of transcriptional initiation
complex
40Structural Motifs in DNA-Binding Regulatory
Proteins
- Crucial feature must be atomic contacts between
protein residues and bases and sugar-phosphate
backbone of DNA - Most contacts are in the major groove of DNA
- 80 of regulatory proteins can be assigned to one
of three classes helix-turn-helix (HTH), zinc
finger (Zn-finger) and leucine zipper (bZIP) - In addition to DNA-binding domains, these
proteins usually possess other domains that
interact with other proteins
41The Helix-Turn-Helix Motif
- contain two alpha helices separated by a loop
with a beta turn - The C-terminal helix fits in major groove of DNA
N-terminal helix stabilizes by hydrophobic
interactions with C-terminal helix
42The Zn-Finger Motif
Zn fingers form a folded beta strand and an alpha
helix that fits into the DNA major groove.
43The Leucine Zipper Motif
- Forms amphipathic alpha helix and a coiled-coil
dimer - Leucine zipper proteins dimerize, either as homo-
or hetero-dimers - The basic region is the DNA-recognition site
- Basic region is often modeled as a pair of
helices that can wrap around the major groove
44Binding of some trans-factors is regulated by
allosteric modification
45Transcription Regulation in Prokaryotes
- Genes for enzymes for pathways are grouped in
clusters on the chromosome - called operons - This allows coordinated expression
- A regulatory sequence adjacent to such a unit
determines whether it is transcribed - this is
the operator - Regulatory proteins work with operators to
control transcription of the genes
46Induction and Repression
- Increased synthesis of genes in response to a
metabolite is induction - Decreased synthesis in response to a metabolite
is repression
47lac operon
- Lac operon encodes 3 proteins involved in
galactosides uptake and catabolism. - Permease imports galactosides (lactose)
- b-galactosidase Cleaves lactose to glucose and
galactose. - b-galactoside transacetylase acetylates
b-galactosides - Expression of lac operon is negatively regulated
by the lacI protein
48The lac I protein
- The structural genes of the lac operon are
controlled by negative regulation - lacI gene product is the lac repressor
- When the lacI protein binds to the lac operator
it prevents transcription - lac repressor 2 domains - DNA binding on
N-term C-term. binds inducer, forms tetramer.
49Inhibition of repression of lac operon by inducer
binding to lacI
- Binding of inducer to lacI cause allosteric
change that prevents binding to the operator - Inducer is allolactose which is formed when
excess lactose is present.
50Catabolite Repression of lac Operon (Positive
regulation)
- When excess glucose is present, the lac operon is
repressed even in the presence of lactose. - In the absence of glucose, the lac operon is
induced. - Absence of glucose results in the increase
synthesis of cAMP - cAMP binds to cAMP regulatory protein (CRP) (AKA
CAP). - When activated by cAMP, CRP binds to lac promoter
and stimulates transcription.
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52Post-transcriptional Modification of RNA
- tRNA Processing
- rRNA Processing
- Eukaryotic mRNA Processing
53tRNA Processing
- tRNA is first transcribed by RNA
- Polymerase III, is then processed
- tRNAs are further processed in the chemical
modification of bases
54rRNA Processing
- Multiple rRNAs are originally transcribed as
single transcript. - In eukaryotes involves RNA polymerase I
- 5 endonuclases involved in the processing
55Processing of Eukaryotic mRNA
565 Capping
- Primary transcripts (aka pre-mRNAs or
heterogeneous nuclear RNA) are usually first
"capped" by a guanylyl group - The reaction is catalyzed by guanylyl transferase
- Capping G residue is methylated at 7-position
- Additional methylations occur at 2'-O positions
of next two residues and at 6-amino of the first
adenine - Modification required to increase mRNA stability
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583'-Polyadenylylation
- Termination of transcription occurs only after
RNA polymerase has transcribed past a consensus
AAUAAA sequence - the poly(A) addition site - 10-30 nucleotides past this site, a string of 100
to 200 adenine residues are added to the mRNA
transcript - the poly(A) tail - poly(A) polymerase adds these A residues
- poly(A) tail may govern stability of the mRNA
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60Splicing of Pre-mRNA
- Pre-mRNA must be capped and polyadenylated before
splicing - In "splicing", the introns are excised and the
exons are sewn together to form mature mRNA - Splicing occurs only in the nucleus
- The 5'-end of an intron in higher eukaryotes is
always GU and the 3'-end is always AG - All introns have a "branch site" 18 to 40
nucleotides upstream from 3'-splice site
61Splicing of Pre-mRNA
- Lariat structure forms by interaction with
5splice site G and 2OH of A in the branch site. - Exons are then joined and lariot is excised.
- Splicing complex includes snRNAs that are
involved in identification of splice junctions.