RNA Synthesis and Processing Transcription Transcriptional Regulation RNA Processing

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RNA Synthesis and ProcessingTranscriptionTranscr
iptional RegulationRNA Processing
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RNA Polymerase and Transcription

• RNA polymerase is the principal enzyme
  responsible for RNA synthesis.
• Like DNA polymerase, RNA polymerase is a complex
  enzyme made up of multiple polypeptide chains.

Figure 7.1 E. coli RNA polymerase
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Transcription in Prokaryotes

• A promoter is the DNA sequence to which RNA
  polymerase binds to initiate transcription of a
  gene.

Figure 7.2 Sequences of E. Coli promoters
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7.3 DNA footprinting

• DNA Footprinting experiments identify sites at
  which RNA polymerase binds to promoters.
• Methods that employ chemical reagents to modify
  and cleave DNA at particular nucleotides can be
  used to identify the specific DNA bases that are
  in contact with protein

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Eukaryotic Transcription and RNA Polymerases

• Eukaryotic cells contain three distinct nuclear
  RNA polymerases that transcribe different classes
  of genes.
• The nuclear RNA polymerases recognize different
  promoters and transcribe distinct classes of
  genes.
• Transcription in eukaryotes takes place on
  chromatin rather than on free DNA.

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General Transcription Factors

• Transcription factors are specific proteins that
  are required for RNA polymerase II to initiate
  transcription.
• The TATA box is a regulatory DNA sequence found
  in the promoters of many eukaryotic genes.
• The TATA-binding protein, or TBP, is a basal
  transcription factor that binds directly to the
  TATA box.
• TBP-associated factors, or TAFs, are polypeptides
  associated with TBP in the general transcription
  factor, TFIID.

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Initiation of Transcription by RNA Polymerase II
Figure 7.12 Formation of a polymerase II
transcription initiation complex
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7.12 Formation of a polymerase II transcription
initiation complex (Part 2)

• Following recruitment of RNA polymerase II to the
  promoter, the binding of two additional
  factorsTFIIE and TFIIHcompletes formation of
  the initiation complex.

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7.14 RNA polymerase II/Mediator complexes

• The Mediator is a large protein complex that
  stimulates basal transcription it also plays a
  key role in linking the general transcription
  factors to the gene-specific transcription
  factors that regulate gene expression.

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Transcription by RNA Polymerases I and II

• RNA polymerase I is devoted solely to the
  transcription of ribosomal RNA genes, which are
  present in tandem repeats.
• The promoters of ribosomal RNA genes span about
  150 base pairs just upstream of the transcription
  initiation site.
• The genes for tRNAs, 5S rRNA, and some of the
  small RNAs involved in splicing and protein
  transport are transcribed by RNA Polymerase III.

Figure 7.15 The ribosomal RNA gene
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Regulation of Transcription in Eukaryotes

• An important difference between transcriptional
  regulation in prokaryotes and eukaryotes results
  from the packaging of eukaryotic DNA into
  chromatin, which limits its availability as a
  template for transcription.
• Noncoding RNAs, as well as proteins, regulate
  transcription in eukaryotic cells via
  modifications in chromatin structure.

Figure 7.18 Identification of eukaryotic
regulatory sequences
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cis-Acting Regulatory Sequences Promoters and
Enhancers

• Certain cis-acting sequences regulate the
  expression of eukaryotic genes.
• Genes transcribed by RNA polymerase II have core
  promoter elements, including the TATA box and the
  Inr sequence, that serve as specific binding
  sites for general transcription factors.

Eukaryotic Promoter
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7.20 The SV40 enhancer

• Enhancers are transcriptional regulatory
  sequences that can be located at a site distant
  from the promoter.
• The activity of enhancers depends on neither
  their distance nor their orientation with respect
  to the transcription initiation site.

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7.21 Action of enhancers
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7.22 DNA looping

• DNA looping allows a transcription factor bound
  to a distant enhancer to interact with proteins
  associated with the RNA polymerase/Mediator
  complex at the promoter
• Enhancers may contain multiple sequence elements
  that bind different transcriptional factor.

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Transcription Factor Binding Sites

• An electrophoretic-mobility shift assay is a
  process in which a radiolabeled DNA fragment is
  incubated with a protein preparation and then
  subjected to electrophoresis through a
  non-denaturing gel.
• The binding sites of most transcription factors
  consist of short DNA sequences, typically
  spanning 610 base pairs.

Figure 7.24 Electrophoretic-mobility shift assay
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Transcription Factor Binding Sites

• Chromatin immunoprecipitation is a method for
  determining regions of DNA that bind
  transcription factors within a cell.

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Structure and Function of Transcriptional
Activators

• Transcriptional activators bind to regulatory DNA
  sequences and stimulate transcription.
• Many different transcription factors contain many
  distinct types of DNA-binding domains.

Figure 7.28 Structure of transcriptional
activators
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7.29 Examples of DNA-binding domains
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Structure and Function of Transcriptional
Activators

• The activation domains of transcription factors
  are not as well characterized as their
  DNA-binding domains.
• Coactivators stimulate transcription by modifying
  chromatin structure.

Figure 7.31 Action of transcriptional activators
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Eukaryotic Repressors

• Many active repressors have been found to play
  key roles in the regulation of transcription in
  animals cells.
• Corepressors act by modifying chromatin
  structure.

Figure 7.32 Action of eukaryotic repressors
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Relationship of Chromatin Structure to
Transcription

• Histone acetylation is the modification of
  histones by the addition of acetyl groups to
  specific lysine residues.
• Like acetylation, certain modifications occur at
  specific amino acid residues in the histone tails.

Figure 7.34 Histone Acetylation
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7.34 Histone acetylation (Part 2)
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Relationship of Chromatin Structure to
Transcription

• Nucleosome remodeling factors are protein
  complexes that alter the arrangement or structure
  of nucleosomes without removing or covalently
  modifying the histones.

Figure 7.36 Nucleosome remodeling factors
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DNA Methylation

• Differences in methylation are maintained
  following DNA replication by an enzyme that
  specifically methylates CpG sequences of a
  daughter strand that is hydorgen-bonded to a
  methylated parental strand.

Figure 7.41 Maintenance of methylation patterns
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Processing of mRNA in Eukaryotes

• Primary transcripts of eukaryotic mRNAs undergo
  extensive modifications before they can serve as
  templates for protein synthesis.
• Pre-mRNA is the primary transcript that is
  processed to form messenger RNA in eukaryotic
  cells.
• A 7-methylguanosine cap is what is added during
  the modification of the 5 end of a transcript.

Figure 7.44 Processing of eukaryotic messenger
RNAs
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7.44 Processing of eukaryotic messenger RNAs
(Part 2)
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Processing of mRNA in Eukaryotes

• A poly-A tail is a tract of about 200 adenine
  nucleotides added to the 3 ends of eukaryotic
  mRNAs.
• Polyadenylation is the process of adding a poly-A
  tail to a pre-mRNA.

Figure 7.45 Formation of the 3 ends of
eukaryotic mRNAs
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7.47 Splicing of pre-mRNA

• In vitro splicing reactions suggest that splicing
  occurs in 2 steps.
• Splicing of pre-mRNA is carried out by large
  complexes called the spliceosomes.

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Splicing Mechanisms

• Small nuclear RNAs, or snRNAs, are nuclear RNAs
  that range in size from 50 to 200 bases.
• Small nuclear ribonucleoprotein particles, or
  snRNPs, are complexes of snRNAs with proteins
  that play central roles in the splicing process.

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Splicing Mechanisms
Figure 7.48 Assembly of the spliceosome
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7.49 Binding of U1 snRNA to the 5 splice site

• The SR splicing factors bind to specific
  sequences within exons and act to recruit U1
  snRNPs to the 5 splice site.

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7.50 Self-splicing introns
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7.51 Role of splicing factors in spliceosome
assembly
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Alternative Splicing

• Alternative splicing is the generation of
  different mRNAs by varying the pattern of
  pre-mRNA splicing.
• In the sex determination of Drosophila,
  alternative splicing of the same pre-mRNA
  determines whether a fly is male or female.

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RNA Degradation

• Most of the sequences transcribed into pre-mRNA
  are degraded within the nucleus.
• Nonsense-mediated mRNA decay is a quality-control
  system that leads to the degradation of mRNAs
  that lack complete open-reading frames.

Figure 7.55 Regulation of transferrin receptor
mRNA stability