Transcription

1
Transcription

• The synthesis of a ribonucleic acid (RNA) polymer
  from a deoxyribonucleic acid (DNA) template
• Separates storage from use
• Provides a control point for regulation
• Amplification step (can make many RNA copies)

RNA
DNA
H
5-TGAGTCACTGTACGCTATATAAGGCGATCGCCTCAGGAACCACCA
TGCT-3 3-ACTCAGTGACATGCGATATATTCCGCTAGCGGAGTC
CTTGGTGGTACGA-5
2
Nucleic acid structure

• Natural DNA adopts a linear double helical form
• complete complementary base-pairing between two
  strands
• RNA transcripts yield complex overall shapes
• synthesized as single strands that fold back upon
  themselves
• folding is driven by base pairing

note GU pair
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General outline of transcription (txn)

• Transcription (Txn) process consists of 5 general
  steps
• BINDING
• RNA polymerase (RNAp) binds to DNA in promoter
  region
• UNWINDING
• Duplex DNA must be unwound to expose bases of
  template strand
• INITIATION
• Polymerize nucleotides one at a time into
  complementary RNA strand
• ELONGATION
• Disengage from additional factors and the
  promoter region to continue transcript synthesis
  throughout full length of gene
• TERMINATION
• Respond to stop signals at the end of the gene
  by stopping synthesis and releasing the RNA
  transcript product

5
General outline of transcription (txn)

• BINDING
• RNA polymerase (RNAp) binds to DNA in Promoter
  region
• Bind to specific sequences or elements
• Additional factors help RNAp recognize promoters
• TXN factors
• General Txn Factors (GTFs) used at all promoters
• Activators/Repressors used at specific promoters
• Bind specific sequence elements
• Directly or indirectly affect RNAp

Co- factor
Txn factor
RNAp
5-TGAGTCACTGTACGCTATATAAGGCGATCGCCTCAGGAACCACCA
TGCT-3 3-ACTCAGTGACATGCGATATATTCCGCTAGCGGAGTC
CTTGGTGGTACGA-5
6
General outline of transcription (txn)

• UNWINDING
• Duplex DNA must be unwound to expose bases of
  template strand
• Helicase enzyme that unwinds duplex regions of
  polynucleotides
• DNA helicases act on DNA strands
• In txn (see below) and DNA replication (not
  covered)
• RNA helicases act on RNA strands
• In a variety of settings, including
  transcriptional regulation

Co- factor
Txn factor
RNAp
CGCCTCAGGAACCA
T
C
5-TGAGTCACTGTACGCTATATAAGGCGA 3-ACTCAGTGACATG
CGATATATTCCGCTAGCGGAGTCCTTGGTGGTACGA-5
CATGCT-3
7
General outline of transcription (txn)

• INITIATION
• Start polymerizing nucleotides one at a time into
  an RNA strand that is complementary to the
  template DNA strand
• Template DNA is read in 3 --gt 5 direction
• The RNA transcript is synthesized in 5 --gt 3
  direction
• RNAn NTP --gt RNAn1 pyrophosphate (PPi)
• PPi --gt 2 inorganic phosphate (Pi)
• A short 10-12nt region of RNA-DNA hybrid is
  created and maintained

Co- factor
Txn factor
RNAp
CGCCTCAGGAACCA
T
C
5-TGAGTCACTGTACGCTATATAAGGCGA 3-ACTCAGTGACATG
CGATATATTCCGCTAGCGGAGTCCTTGGTGGTACGA-5
CATGCT-3
GCCUCAGGAT-3
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General outline of transcription (txn)

• ELONGATION
• Disengagement of RNAp from various GTFs,
  cofactors and the promoter region is often called
  Promoter Escape
• After disengaging, RNAp continues transcript
  synthesis throughout full length of gene
• A short 10-12nt region of RNA-DNA hybrid is
  maintained
• Transcribed region of DNA is allowed to re-anneal
  (close), displacing the RNA strand
• RNAp moves 3 --gt 5 on the DNA template strand
• Note movement is 5 --gt 3 on the non-template
  DNA strand

Co- factor
Txn factor
RNAp
ACCACCATGCT-3
A
5-TGAGTCACTGTACGCTATATAAGGCGATCGCCTCAGG 3-ACT
CAGTGACATGCGATATATTCCGCTAGCGGAGTCCTTGGTGGTACGA-5

ACCACCAUGC-3
GCCUCAGGA
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General outline of transcription (txn)

• TERMINATION
• Respond to stop signal sequences indicating the
  end of the gene
• stop synthesis (a pause in the polymerization
  reaction)
• release the RNA transcript product
• Release RNAp from the DNA

RNApII
CAATAAACTAAATTATTA
A C
5-CCATGCT CTTATGTACGTAGCGACT
-3 3-GGTACGATGTTATTTGATTTAATAATGGAATACATGCATCGC
TGA-5
AGAAUAAACU-3
CCAUGCU
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Bacterial txn

• RNAp core enzyme
• 5 subunits capable of binding DNA RNA synthesis
• No promoter specificity
• Sigma factors target RNAp to different types of
  promoters
• Sigma70 is for housekeeping and most other
  genes
• Genes always transcribed (glycolysis enzymes,
  etc), many inducible too
• -35 element TTGACA
• -10 element TATAAT
• Sigma32 is for heat shock genes
• Chaperone genes induced in response to excess heat

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• BACTERIA
• BIND
• UNWIND
• INITIATE
• ELONGATE
• without sigma

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• BACTERIA
• TERMINATE
• Rho-dependent
• Rho protein (helicase) unwinds RNA-DNA duplex
  causing release of finished RNA transcript
• Rho-independent
• Rho protein is not required
• DNA terminator sequence causes RNAp to pause,
  release from DNA and release RNA transcript

Rho
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Txn in eukaryotes

• Three different RNAp enzymes RNApI, RNApII,
  RNApIII
• All eukaryotic RNAs require additional processing
  steps after synthesis to yield the mature RNA
• Primary RNA transcripts are often called
  pre-RNAs

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Txn in eukaryotes RNApI

• 100s of copies of the large ribosomal RNA (rRNA)
  genes in most genomes
• Large numbers needed to yield large amounts of
  RNA
• Copies are grouped into clusters called rDNA
• For example, humans have 5 rDNA clusters
• rDNA clusters are grouped within the nucleus to
  form the nucleoli
• Nucleoli are the site of rRNA synthesis,
  processing and ribosome assembly

18S 5.8S 28S
18S 5.8S 28S
18S 5.8S 28S
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Txn in eukaryotes RNApI

• RNApI molecules, densely packed on DNA template
• Very high rate of rRNA synthesis
• pre-rRNA must be processed to yield mature rRNA

18S 5.8S 28S
18S 5.8S 28S
18S 5.8S 28S
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Txn processing in eukaryotes RNApI

• pre-rRNA processing requires small nucleolar RNAs
  (snoRNAs)
• snoRNAs exist in complexes with proteins to yield
  various snoRNPs
• Some are involved in the cleavage and trimming
  reactions
• Separate the 18S rRNA from the 5.8S rRNA
• Separate the 5.8S rRNA from 28S rRNA

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Txn processing in eukaryotes RNApI

• snoRNPs also direct chemical modifications to
  rRNAs
• snoRNA sequence grants specificity through base
  pairing with rRNA
• Base modifications, (e.g. pseudouridine)
• Ribose modification (e.g. 2-OH methylation)
• Increase RNA stability

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Txn in eukaryotes RNApIII

• RNApIII can recognize as promoter sequences,
  regions internal to the transcription unit
• Specific General Transcription Factors (GTFs)
  enable promoter binding
• e.g. TFIIIA, TFIIIC
• (Note, there are GTFs for RNApI also, TFIA,
  etc)

GTF
RNAp
CGCCTCAGGAACCA
T
C
5-TACGCTGTCTAGGCGA 3-ATGCGACAGATCCGCTAGCGGAGTC
CTTGGTGGCATAGGAGTTAGGGA-5
CGTATCCTCAATCCCT-3
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Txn in eukaryotes RNApIII

• Transcribe 5S rRNA genes
• Product transported to nucleoli for
  processing/assembly into ribosomes
• Transcribe tRNA genes
• Exist in genomic clusters
• Clusters contain a variety of different tRNAs
• Total number of tRNA genes can be very high
• (e.g. 275 in yeast, 1300 in humans)
• Primary tRNA transcripts require processing to
  mature form
• Processing involves cutting and trimming
  reactions
• Various ribonuclease enzymes required (RNAse P,
  etc)

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Txn in eukaryotes RNApII

• Transcribe messenger RNA (mRNA) and microRNA
  (miRNA) genes
• RNApII
• 12 subunits core enzyme
• Promoter specificity requires 6 additional GTFs
• TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH
• TATA box, at -24 to -32 matches closely to
  TATATAA
• TFIID contains the TATA Binding Protein (TBP)

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• RNApII txn
• BIND
• TFIID, via TBP, binds TATA box DNA sequence
• TFIIA TFIIB add next, assemble with some DNA
  sequence selectivity
• A TFIIF-RNApII complex binds
• TFIIE TFIIH bind to complete the
  pre-initiation complex

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• RNApII txn
• UNWIND
• TFIIH contains a helicase subunit for unwinding
  the promoter region
• INITIATE
• Synthesize 10-12nts
• ELONGATE
• TFIIH contains two kinase subunits that
  phosphorylate the C-terminal Domain (CTD) of
  RNApII
• CTD repeat YSPTSPS
• Breaks contacts w/ promoter

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RNApII transcript processing

• mRNA structure
• 5-end is capped
• Capping enzymes bind to phospho-CTD of RNApII
• Unusual 5 -- 5 linkage of 7-methylG-PPP
• Protects 5-end from exonucleases
• Enhances nuclear export
• Enhances mRNA tln

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RNApII transcript processing

• mRNA structure
• 5-untranslated region (UTR)
• Regulation of translation (TLN) and stability
• Coding region
• Sequence of nucleotides continuously encoding a
  protein
• Also called an open reading frame (ORF)
• 3-UTR
• Regulation of translation (TLN) and stability

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• mRNA structure
• 3-end is polyadenylated
• Requires a large protein complex
• (e.g. CPSF, CStF)
• Recognizes 5-AAUAAA-3 sequence in primary
  transcript
• Cleaves pre-RNA 20nt downstream of AAUAAA
• PolyA polymerase adds 50-250 adenosines at new
  3-end
• Protects 3-end from exonucleases
• TERMINATE
• Recognition of AAUAAA coupled with RNApII
  destabilization
• Cleavage effectively releases pre-RNA from RNApII
• RNApII with reduced processivity falls off DNA
  template

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RNApII transcript processing

• Primary transcripts for protein coding genes
  (hnRNAs) are much larger than their corresponding
  mRNAs
• Heteronuclear RNAs
• Localized to the cell nucleus
• Contain exon sequences
• Contain intron sequences
• mRNAs
• Localized to the cell cytoplasm
• Contain only exon sequences
• RNA splicing
• Removal of introns
• Joining exons together

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RNApII transcript processing splicing

• Exons can be either protein coding or 5-/3-UTR
  sequences
• Exons contribute to the final mRNA product
• 150nts each
• Intervening sequences between exons are introns
• Introns must be removed to yield the final mRNA
  product
• 3500nts each

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RNApII transcript processing splicing

• Specific RNA sequences demarcate the exon/intron
  borders
• Subunits of the spliceosome recognize these
  exon junctions

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• The spliceosome catalyzes two reactions that
  eliminate the intron and join upstream and
  downstream exons together

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• Spliceosomes are assembling on pre-RNA during
  Elongation
• Provides mechanism to avoid confusing which exons
  go together

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• Spliceosomes are assembling on pre-RNA during
  Elongation
• Provides mechanism to avoid confusing which exons
  go together
• Sequential assembly of spliceosomes as pre-RNA is
  synthesized helps assure no exon/intron junctions
  are accidentally missed
• Which ones should be made?

Exon 1
Exon 2
Exon 3
Exon 4
Exon 1
Exon 2
Exon 3
Exon 4
Exon 1
Exon 3
Exon 4
Exon 1
Exon 2
Exon 4
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Alternative splicing

• Not all exons have ideal exon/intron splicing
  sequences
• Not all are efficiently recognized by spliceosome
• Exonic Splicing Enhancer (ESE) sequences
• Binding factors can promote use of an exon/intron
  junction
• - ESE binding protein
• ESE binding protein

Exon 1
ESE
Exon 3
Exon 4
Exon 1
Exon 3
Exon 4
Exon 1
ESE
Exon 3
Exon 4