Transcription - PowerPoint PPT Presentation

About This Presentation
Title:

Transcription

Description:

'Housekeeping' genes encode proteins or RNAs that are essential for normal cellular ... Leads to disassembly of RNA polymerase complex. Rho Dependent Termination ... – PowerPoint PPT presentation

Number of Views:85
Avg rating:3.0/5.0
Slides: 62
Provided by: davidsh5
Category:

less

Transcript and Presenter's Notes

Title: Transcription


1
  • Transcription

2
Central Dogma
3
Genes
  • 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.

4
Types 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

5
RNA 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
6
Phases 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.

7
E. 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
8
s-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

9
General 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.

10
Gene Promoters
  • Site where RNA polymerase binds and initiates
    transcription.
  • Gene that are regulated similarly contain common
    DNA sequences (concensus sequences) within their
    promoters

11
Important Concensus Sequences
  • Pribnow Box position 10 from transcriptional
    start
  • -35 region position 35 from transcriptional
    start.
  • Site where s70-factor binds.

12
Other s-Factors
  • Standard genes s70
  • Nitrogen regulated genes s54
  • Heat shock regulated genes s32

13
How 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

14
Transcriptional 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).

15
Initiation 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.

16
Transcriptional Initiation
Closed complex
Open complex
Primer formation
Disassociation of s-factor
17
Chain 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

18
(No Transcript)
19
Transcriptional Termination
  • Process by which RNA polymerase complex
    disassembles from 3 end of gene.
  • Two Mechanisms Pausing and rho-mediated
    termination

20
Pausing 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
21
Rho Dependent Termination
  • rho is an ATP-dependent helicase
  • it moves along RNA transcript, finds the
    "bubble", unwinds it and releases RNA chain

22
(No Transcript)
23
Eukaryotic Transcription
  • Similar to what occurs in prokaryotes, but
    requires more accessory proteins in RNA
    polymerase complex.
  • Multiple RNA polymerases

24
Eukaryotic 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
25
Eukaryotic 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

26
Eukaryotic Gene Promoters
  • Contain AT rich concensus sequence located 19 to
    27 bp from transcription start (TATA box)
  • Site where RNA polymerase II binds

27
RNA 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

28
More 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

29
Transcription 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

30
Transcription 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

31
Eukaryotic 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

33
Gene Expression
  • Constitutive Genes expressed in all cells
    (Housekeeping genes)
  • Induced Genes whose expression is regulated by
    environmental, developmental, or metabolic
    signals.

34
Regulation of Gene Expression
RNA Processing
mRNA
RNA Degradation
AAAAAA
5CAP
Active enzyme
Post-translational modification
Protein Degradation
35
Transcriptional 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

36
Cis-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.

37
Enhancers
  • 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

38
(No Transcript)
39
Two 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

40
Structural 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

41
The 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

42
The Zn-Finger Motif
Zn fingers form a folded beta strand and an alpha
helix that fits into the DNA major groove.
43
The 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

44
Binding of some trans-factors is regulated by
allosteric modification
45
Transcription 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

46
Induction and Repression
  • Increased synthesis of genes in response to a
    metabolite is induction
  • Decreased synthesis in response to a metabolite
    is repression

47
lac 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

48
The 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.

49
Inhibition 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.

50
Catabolite 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.

51
(No Transcript)
52
Post-transcriptional Modification of RNA
  • tRNA Processing
  • rRNA Processing
  • Eukaryotic mRNA Processing

53
tRNA Processing
  • tRNA is first transcribed by RNA
  • Polymerase III, is then processed
  • tRNAs are further processed in the chemical
    modification of bases

54
rRNA Processing
  • Multiple rRNAs are originally transcribed as
    single transcript.
  • In eukaryotes involves RNA polymerase I
  • 5 endonuclases involved in the processing

55
Processing of Eukaryotic mRNA
56
5 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

57
(No Transcript)
58
3'-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

59
(No Transcript)
60
Splicing 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

61
Splicing 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.
Write a Comment
User Comments (0)
About PowerShow.com