Transcript ppt

7 RNA Synthesis and Processing
Chapt. 7 RNA synthesis and processing brief
Student learning outcomes:
1. Explain transcription in Prokaryotes
2*. Explain how Eukaryotic transcription is complex:
RNA Polymerases, General Transcription Factors
3. Briefly explain complex regulation of transcription in
Eukaryotes: many proteins
4*. Describe RNA processing: Cap, polyA, splicing
5. Describe RNA turnover, Nonsense-mediated decay
Introduction
Regulation of gene expression allows cells:
• to adapt to environmental changes
• is responsible for distinct activities of differentiated
cell types that make up complex organisms.
Transcription is synthesis of primary RNA from gene.
• first step in gene expression (+1 = start of RNA)
• initial level at which gene expression is regulated.
RNAs in eukaryotic cells are modified and processed
Gene structure:
promoter, +1, 5’-UTR, coding region, 3’-UTR, terminator
Transcription in Prokaryotes
1. Prokaryotes:
E. Coli RNA polymerase (RNAP) polymerizes
ribonucleoside 5′-triphosphates (NTPs), 5′ to 3′
•
directed by DNA template
Bacterial RNAP has 5 types of subunits.
•
•
Sigma (σ) subunit is weakly bound; required to identify
correct sites (promoter) for initiation.
Different s can direct RNAP to different start sites under
different conditions
Fig. 7.1
Fig. 7.5
Crab-claw shape
Active site channel
Transcription in Prokaryotes
Promoter - gene sequence to which RNAP binds to
initiate transcription.
• Prokaryote promoters: 6 conserved nucleotides 10
and 35 bp upstream of transcription start site (+1).
• Consensus sequences - bases most frequently
found in different promoters.
Transcription initiates de novo (no primer required)
•
major step for regulation of gene expression
Fig. 7.2 prokaryotic promoter
Fig 7.3 DNA footprinting
DNA footprinting
identifies sites RNAP binds:
DNA fragment labeled one end
(radioisotope or fluorescent dye)
Incubated with RNAP,
Partially digest with DNase.
Bound protein protects DNA from
DNase digestion,
Identify sequences compared to
DNA with no protein.
Fig. 7.3
Fig 7.4 Transcription by E. coli RNA polymerase
Initiation: σ binds
specifically to –35 and –10
sequences, starting
transcription
Initial binding is
closed-promoter
complex because
DNA is not unwound
During elongation,
RNAP maintains
unwound region of
15 base pairs.
Fig 7.6 Transcription termination
Termination: RNA synthesis continues until RNAP
encounters termination signal.
• Common signal is symmetrical inverted repeat of GC-rich
sequence followed by seven A residues
• Transcription of GC-rich inverted repeat results in segment of
RNA that can form stable stem loop structure.
•
disrupts association
with DNA template,
terminates transcription
Fig. 7.6
Transcription in Prokaryotes
Prokaryotic gene regulation:
• control of transcription by interaction of regulatory
proteins with specific DNA sequences
Cis-acting control elements only affect expression of
linked genes on same DNA molecule (e.g. operator).
Trans-acting factors can affect expression of genes
located on other chromosomes (e.g. repressor).
lac operon is an example of negative control—binding of
repressor blocks transcription.
lac operon is an example of positive control—binding of cAMP
to CAP protein enhances transcription
.
Fig 7.8 Negative control of the lac operon
lac operon: negative regulation of transcription:
Enzymes metabolize lactose
Z is b-galactosidase gene;
Repressor (i) binds O and
blocks transcription;
Presence of lactose binds
to allosteric site on R,
R is inactive;
transcription starts
Fig. 7.8
Fig 7.9 Positive control of the lac operon by glucose
lac operon: Positive control
of transcription in E. coli :
Presence of glucose
(preferred energy source)
represses expression of genes
for enzymes that break down
other sugars, such as lac operon
Low glucose activates synthesis
of cAMP, binds CAP protein,
activates expression of operon
(still need lactose inactivate R)
Fig. 7.9
2. Eukaryotic cells
have 3 nuclear RNAP
transcribe different
classes of genes:
Pol I, Pol II, Pol III
Pol II makes mRNA
• Each has12 to 17 different
subunits.
• 9 conserved subunits,
5 of which are related to
subunits of bacterial RNAP
Eukaryotic RNA Polymerases and General Transcription Factors
Pol II synthesizes mRNA
• requires initiation factors not associated with RNAP
• Structure by Roger Kornberg – Nobel Prize
General transcription factors (GTFs) - proteins
involved in transcription from pol II promoters
• 10% of human genes transcription factors (GTF, regulatory)
• Promoters have different regulatory sequences.
Fig. 7.10 pol II
Fig 7.11 Formation of a polymerase II preinitiation complex in vitro
Pol II Promoter sequence elements include TATA box
• (resembles –10 sequence element of bacterial promoters)
Minimum of 5 GTFs required for in vitro initiation of transcription.
• TFIID (TBP + TAFs), TFIIB, TFIIF, RNAP, TFIIE, TFIIH,
Fig 7.13 RNA polymerase II/Mediator complexes and transcription initiation
Additional factors required to initiate transcription:
• Mediator, large protein complex of more than 20 subunits
• interacts with both GTFs and pol II
CTD of pol II is
phosphorylated,
binds other factors
that assist processing,
elongation, termination
Fig. 7.13
Fig 7.20 Regulation: Action of enhancers
cis-acting sequences
regulate expression
Transcription factors
bind upstream DNA
sequences (enhancers)
• Activity of enhancers doesn’t
depend on distance,
orientation to initiation site.
Fig. 7.20 enhancer sequences
Fig 7.21 DNA looping
Enhancers, like promoters, bind transcription factors
that regulate pol II
DNA looping allows transcription factor bound to distant
enhancer to interact with proteins associated with pol II
/Mediator complex at promoter
DNA is bound in chromatin: (nucleosomes wrap DNA)
modifications of chromatin
structure affect regulation
Fig. 7.21
Fig 7.23 Technique: Electrophoretic-mobility shift assay
EMSA (Electrophoretic-mobility shift assay)
identifies transcription factor binding sites in vitro
Radiolabeled DNA fragments incubated with protein,
then electrophoresed through nondenaturing gel.
Migration of DNA fragment through gel slowed by bound protein
Binding sites are usually
short DNA sequences
(6–10 base pairs),
and are degenerate.
Fig. 7.23
Fig 7.25 Technique: Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) identifies
DNA regions that bind transcription factors in vivo.
• Cross-link TF to their specific DNA sequences
• Chromatin is extracted, fragmented. Immunoprecipitation
isolates fragments of DNA linked to TF
• PCR identifies fragments
Fig. 7.25
Fig 7.27 transcriptional activators
Transcriptional activators bind to regulatory DNA
sequences and stimulate transcription.
Factors have two independent domains:
• one binds DNA (common motifs),
• other stimulates transcription by interacting with other
proteins (such as Mediator, pol II, coactivators)
Less common, gene expression regulated by repressors bind DNA sequences, inhibit transcription
Fig. 7.27
Fig. 7.31
RNA Processing and Turnover
4. RNA processing and turnover:
Primary transcript of gene is only the first step:
Most newly-synthesized RNAs must be modified,
except bacterial mRNAs which are used immediately for
protein synthesis while still being transcribed.
rRNAs and tRNAs must be processed in both
prokaryotic and eukaryotic cells.
Regulation of processing provides another level of
control of gene expression.
Fig 7.43 Processing of ribosomal RNAs
rRNAs derive from long pre-rRNA molecule
• Pol I synthesizes eukaryotic rRNAs (tandem repeat genes)
• 5S rRNA in eukaryotes is transcribed from separate gene.
Fig. 7.43
Fig 7.44 Processing of transfer RNAs (Part 1)
tRNAs start as pre-tRNAs, (prokaryotes and eukaryotes)
• Processing of 5′ end of pre-tRNAs - cleavage by RNase P.
• RNase P is ribozyme: RNA has catalytic activity
• Processing of 3′ end of tRNAs: cleavage by nuclease,
•
addition of CCA terminus, (site of aa attachment)
•
Some bases modified.
Fig. 7.44
RNA Processing and Turnover
**Eukaryote pre-mRNAs extensively modified before
export from nucleus:
• 5’-CAP, splicing, 3’-cleavage/polyA addition
Transcription and processing are coupled.
C-terminal domain (CTD) of RNA pol II plays key role
in coordinating processes:
• After phosphorylation, binds other protein complexes:
•
•
Capping enzymes bind phosphorylated CTD after initiation
Cap added after transcription of first 20 to 30 nucleotides
Fig 7.45 Processing of eukaryotic messenger RNAs
5′ end of transcript modified by addition
of 7-methylguanosine cap
Fig. 7.45
Fig 7.46 Formation of 3´ ends of eukaryotic mRNAs
• At 3′ end, poly-A tail is added by polyadenylation.
• Signals for cleavage, polyadenylation include:
•
conserved hexanucleotide (AAUAAA in mammalian cells),
•
G-U rich downstream sequence element.
• Pol II not recognize termination sequences:
Fig. 7.46
•
stops after mRNA cleaved.
Fig 7.48 Splicing of pre-mRNA
**Splicing: Introns (noncoding) removed from pre-mRNA
Splicing proceeds in 2 steps: (lariat intermediate)
• 1. Cleavage at 5′ splice site (SS), joining 5′ end of intron to A
within intron (branch point). Intron forms loop.
• 2. Cleavage at 3′ splice site, ligation of exons, excision intron
Important sequences:
• 5’ splice site
• 3’ splice site
• Branch point
In vitro experiments
(gel electrophoresis,
mutant sequences)
Fig. 7.47
RNA Processing and Turnover
Spliceosomes: large complexes that splice:
• 5 types of small nuclear RNAs (snRNAs)—
U1, U2, U4, U5, and U6.
• 6-10 protein molecules
small nuclear ribonucleoprotein
particles (snRNPs); RNAs are catalytic
• Classic expt by Joan Steitz identified snRNPs:
antibody from Lupus patients: antigens SM, RNP
32P-cells; IP proteins, analyze RNAs
1: anti-Sm; 2, normal; 3, anti-RNP, 4, anti-RNP
• X is nonspecific band
Fig 7.49 Assembly of the spliceosome
Spliceosome assembly:
1. U1 snRNP binds to 5′ SS (complementary base pairs)
2. U2 snRNP binds to branch point
3. Other snRNPs join complex, form intron loop
4. Maintain association of 5′ and 3′ exons: so can be ligated
followed by excision of intron.
Fig. 7.49
Fig 7.51 Self-splicing introns
Some RNAs self-splice: catalyze removal of own
introns (absence of other protein or RNA factors).
– Group I — cleavage at 5′ SS mediated by G cofactor. The 3′
end of free exon reacts with 3′ SS to excise intron.
– Group II—cleavage of 5′ SS results from attack by A in
intron, results in lariat-like intermediate, which is excised.
Fig. 7.51
Fig 7.52 Splicing factors assist spliceosome assembly
Other splicing factors bind specific RNA sequences
and recruit U1 and U2 snRNPs to appropriate sites
•
•
SR factors bind specific sequences in exons,
• recruit U1 snRNP to 5′ SS.
U2AF binds pyrimidine-rich sequences at 3 ′ SS
• recruits U2 snRNP to branch point.
Fig. 7.52
RNA Processing and Turnover
*Alternative splicing occurs in complex eukaryotes
•
Most pre-mRNAs contain multiple introns: different mRNAs
can be produced from same gene.
• Novel means of controlling gene expression,
• Increases diversity of proteins that can be encoded
Sex determination Drosophila; tissue-specific splicing.
• Alternative splicing of transformer mRNA regulated by SXL
protein, only expressed in female flies.
• SXL repressor blocks splicing factor U2AF.
Fig. 7.53
Alternative splicing
Ex. Dscam gene of Drosophila encodes cell surface
protein: connections between neurons in fly brain
• 4 sets of alternative exons;
• one from each set goes into spliced mRNA.
• Exons joined in any combination: alternative splicing
potentially yield 38,016 different mRNAs.
Fig. 7.54
RNA Processing and Turnover
RNA editing: processing (not splicing) that alters
protein-coding sequences of some mRNAs.
• Single base modifications: deamination of C to U, A to I
Ex. Editing mRNA for apolipoprotein B, (transports lipids in
blood), results in two different proteins:
• Apo-B100 (4536 aa) synthesized in liver (unedited mRNA)
• Apo-B48 (2152 aa) synthesized in intestine, edited mRNA
• (a C changed to U by deamination created stop codon)
.
Fig. 7.55
RNA Processing and Turnover
Aberrant mRNAs can be degraded.
Nonsense-mediated mRNA decay eliminates mRNAs
that lack complete open-reading frames.
•
•
•
Ribosomes encounter premature termination codons,
translation stops, defective mRNA degraded
Ultimately, RNAs degraded in cytoplasm.
Rate of degradation controls gene expression
rRNAs and tRNAs very stable, (both prokaryotes, eukaryotes)
• high levels of these RNAs (greater than 90% of all RNA)
Bacterial mRNAs rapidly degraded: t1/2 2 to 3 minutes.
• Rapid turnover lets cell respond quickly to changes in
environment, such as nutrient availability
.
RNA Processing and Turnover
Eukaryotic mRNA half-lives vary; < 30 min to 20 hrs
• Short-lived mRNAs encode regulatory proteins:
levels can vary rapidly in response to environmental stimuli.
• mRNAs encoding structural proteins or central
metabolic enzymes longer half-lives
•
Degradation of mRNAs initiated by shortening poly-A tails.
Fig. 7.56
Review questions
1. How does footprinting identify protein binding sites?
6. How do enhancers differ from promoters as cis-acting
regulatory sites
14. How are 2 structurally and functionally different forms of
apolipoprotein B synthesized in liver and intestine?
15. What is nonsense-mediated mRNA decay and its
significance?