Ch8 Transcription Slides1-30 eLC

You should read/review Chapter 1.3 on your You should read/review Chapter 1.3 on your ownown

8.1 RNA transcripts carry the messages of genes

8.2 Bacterial transcription is a four-stage process

8.3 Eukaryotic transcription uses multiple RNA polymerases

8.4 Post-transcriptional processing modifies RNA molecules

1GENE3200 – Bedell – Chapter 8 – Oct 8, 2012

An electron micrograph of splicesosomes engaged in intron splicing

The central dogma of biologyThe central dogma of biology

GENE3200 – Bedell – Chapter 8 – Oct 8, 2012 2

Sanders & BowmanFig. 1.8

RNA compositionRNA composition

Ribonucleotides are composed of a sugar, nucleotide base, and one or more phosphate groups

The bases are adenine, guanine, cytosine, and uracil (NO thymine)

The sugar is ribose (NOT deoxyribose)


Comparison of DNA and RNAComparison of DNA and RNA

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Characteristic DNA RNA

Bases G, A, T, C G, A, U, C

Sugar Deoxyribose Ribose

Strands Double-stranded Single-stranded

Base-pairing G:C, A:T G:C, A:U, G:U

GENE3200 – Bedell – Chapter 8 – Oct 8, 2012

Comparison of DNA replication and transcriptionComparison of DNA replication and transcription

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Characteristic Replication Transcription

Enzyme DNA polymerase RNA polymerase

Primer needed Yes No

Site of initiation Origin (Ori) Promoter

Synthesis direction 5’ 3’ 5’ 3’

Site of termination Chromosome end Termination site

Product ds DNA ss RNA


RNA classificationRNA classification

Messenger RNAs (mRNAs) encode polypeptides

Functional RNAs [also called non-coding RNAs (ncRNAs)] - are not translated but have important structural functions

• Required for polypeptide synthesis

Transfer RNA (tRNA)

Ribosomal RNA (rRNA)

• Required for RNA processing (eukaryotes only)

Small nuclear RNA (snRNA)

6GENE3200 – Bedell – Chapter 8 – Oct 8, 2012

RNA classification (cont)RNA classification (cont)

Functional RNAs [also called non-coding RNAs (ncRNAs)] that are not translated but have important regulatory roles

• Posttranscriptional regulation of gene expression (eukaryotes only)

MicroRNA (miRNA) – discussed in Chapter 15

•Protect genomes from viruses and transposons (eukaryotes only)

•Small interfering RNA (siRNA) – discussed in Chapter 12

•Catalytic activity (eukaryotes only)

•Ribozymes


The product of transcription is a single-stranded The product of transcription is a single-stranded primary transcriptprimary transcript

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For a given ds DNA sequence, unless the template strand or the direction of transcription is stated, either strand of DNA could be the template for transcription

3'

5'

5'

3'

DNA

Transcription

RNA

5'

3' Primary transcript

Template strand

RNA-like RNA-like strandstrand

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Know how to answer questions like theseKnow how to answer questions like these

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If this is the RNA-like (coding strand), what is the sequence of its RNA transcript?

For the RNA sequence: 5’-GUCCA-3’

What is the sequence of its RNA-like (coding) strand of DNA?

What is the sequence of its template (noncoding) strand of DNA?

For the DNA sequence: 5’-CTAGAT-3’

What is the sequence of its complementary DNA strand?3’-GATCTA-5’

5’-CUAGAU-3’

3’-CAGGT-5’

5’-GTCCA-3’


General diagram of a geneGeneral diagram of a gene

The promoter is a DNA sequence that determines where transcription initiates but isn’t part of the transcribed sequence

RNA polymerase binds to the promoter and transcription initiates at a defined distance from the promoter

The transcription start site is designated +1


Bacterial RNA polymeraseBacterial RNA polymerase

The core enzyme cannot bind the promoter or initiate RNA synthesis without the subunit

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++ ==

Bacterial promoter structureBacterial promoter structure

RNA polymerase binds to 10 and 35 sequences

10 position (Pribnow box), consensus is 5-TATAAT-3

35 position, consensus sequence is 5- TTGACA-3


Transcription initiation in prokaryotesTranscription initiation in prokaryotes

RNA polymerase holoenzyme binds to the promoter (-10 and -35 sequences) to form the closed promoter complex

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Approx. 18 bp of DNA around the 10 position to form the open promoter complex

Transcription elongation and termination in Transcription elongation and termination in prokaryotesprokaryotes

After initiation holoenzyme synthesizes 8 – 10 nt of RNA, then sigma subunit dissociates from the core enzyme

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Synthesis by core enzyme occurs until a termination sequence is encountered

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Termination of transcription in prokaryotesTermination of transcription in prokaryotes

When RNA polymerase reaches the termination sequence, the core enzyme dissociates from the DNA and the RNA transcript is released

Shortly after one round of transcription is initiated, a second round begins


Two transcription termination mechanisms in Two transcription termination mechanisms in prokaryotesprokaryotes

Termination of transcription in bacteria is signaled by a DNA termination sequence that usually contains a repeating sequence

In intrinsic termination, a mechanism dependent only on the presence of the repeat induces secondary structure needed for termination

rho-dependent termination requires a different termination sequence and the rho protein


Transcription termination in prokaryotesTranscription termination in prokaryotes

Each transcript of a gene terminates at the same site

Two kinds of terminators in prokaryotes:

• Rho-dependent termination

Requires Rho (ρ) factor

RNA polymerase pauses, then ρ factor mediates dissociation of RNA from RNA polymerase

• Intrinsic terminator

Sequences consist of inverted repeat that forms a hairpin structure (intramolecular hydrogen bonding)

3' end of RNA is usually 10-20 nt downstream to terminator

Used for termination in most prokaryotic genes


Intrinsic termination in prokaryotesIntrinsic termination in prokaryotesG

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Intrinsic Intrinsic termination in termination in prokaryotes prokaryotes

(cont)(cont)

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Inverted repeat sequences in mRNA form a stem-loop structure (hairpin)

Series of U’s in the mRNA causes RNA polymerase to slow down and destabilize

The instability caused by the slowing polymerase and the U-A base pairs induces the polymerase to release the transcript and separate from the DNA GENE3200 – Bedell – Chapter 8 – Oct 8, 2012

Rho-dependent terminationRho-dependent termination

Rho protein recognizes the rho utilization (or rut) site, a stretch of about 50 nucleotides rich in cytosines

Rho then moves along the transcript to RNA polymerase and catalyzes the breakage of hydrogen bonds between the mRNA and the DNA template, and release of the polymerase


In eukaryotes, transcription is more complex In eukaryotes, transcription is more complex than in prokaryotesthan in prokaryotes

Eukaryotic promoters are more diverse than those of bacteria

The complex that assembles to initiate and elongate transcription is more complicated than in bacteria

Eukaryotic genes carry introns and exons, and require processing to remove introns

Eukaryote DNA is associated with proteins to form chromatin

• The chromatin structure affects transcription and plays an important role in gene regulation of eukaryotes


Eukaryotic transcription uses multiple RNA Eukaryotic transcription uses multiple RNA polymerasespolymerases

Eukaryotes have three different RNA polymerases that recognize different promoters and produce different types of RNAs

• RNA polymerase I (RNA pol I) transcribes three ribosomal RNA genes

• RNA polymerase II (RNA pol II) transcribes protein coding genes and most small nuclear RNA genes

• RNA polymerase III (RNA pol III) transcribes tRNA, one small nuclear RNA, and one ribosomal RNA


RNA polymerase II promoter sequences in RNA polymerase II promoter sequences in eukaryoteseukaryotes

The most common eukaryotic promoter consensus sequence is the TATA box, or the Goldberg-Hogness box, located at about position 25

• The consensus sequence is 5-TATAAA-3

A CAAT box is often found near the -80 position

A GC-rich box (consensus 5-GGGCGG-3) is located at 90, or further upstream

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Promoter sequences in eukaryotes (cont)Promoter sequences in eukaryotes (cont)

Eukaryotic promoters display a high degree of variability in type, number, and location of consensus sequence elements

The TATA box is most common, whereas the CAAT box and GC-rich box are more variable

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Promoter recognition in eukaryotesPromoter recognition in eukaryotes

RNA pol II recognizes and binds to promoter sequences with the aid of proteins called transcription factors (TFs)

TFs bind to regulatory sequences and interact directly, or indirectly, with RNA polymerase

• TFs interacting with pol II are called TFII factors

The TATA box is the principle binding site during promoter recognition


Transcription initiation in eukaryotesTranscription initiation in eukaryotes


TFIID consists of the TATA-binding protein (TBP) and a protein called the TBP-associated factor (TAF)

The assembled TFIID binds to the TATA box and forms the initial committed complex

TFIIB, TFIIF, and RNA pol II join the complex to form the minimal initiation complex

Transcription initiation in eukaryotes (cont)Transcription initiation in eukaryotes (cont)

The minimal initiation complex is joined by TFIIE and TFIIH to form the complete initiation complex

The complete initiation complex contains multiple proteins called “general transcription factors”

The complete complex directs RNA pol II to the 1 position, where it begins to assemble mRNA


Enhancers and silencers lead to differential Enhancers and silencers lead to differential expression of eukaryotic genesexpression of eukaryotic genes

Promoters alone are not sufficient to initiate transcription of many eukaryotic genes

Enhancer sequences

• Increase the level of transcription of specific genes

• Activator proteins and their cofactors bind to enhancers

Silencer sequences

• Repress transcription of specific genes

• Repressor proteins bind to silencers

Enhancers and silencers may be located variable distances from their target genes, either upstream or downstream

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Enhancer sequences and DNA bendingEnhancer sequences and DNA bending

Activator proteins and their coactivators form a “protein bridge” that links the proteins at the enhancer sequence to the initiation complex at the promoter


This bridge bends the DNA so that the proteins at both locations are brought close enough together for them to interact

Silencer sequencesSilencer sequences

Silencer sequences are DNA elements that act at a distance to repress transcription of their target genes

Silencers bind transcription factors called repressor proteins that induce bends in DNA

These bends reduce transcription of the target gene

Silencers may be located variable distances from their target genes, either upstream or downstream


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Ch8 Transcription Slides1-30 eLC