Ch8 Transcription Slides1-30 eLC

You should read/review Chapter 1.3 on your own

8.1 RNA transcripts carry the messages of genes 8.2 Bacterial transcription is a fourstage process 8.3 Eukaryotic transcription uses multiple RNA polymerases 8.4 Post-transcriptional processing modifies RNA moleculesAn electron micrograph of splicesosomes engaged in intron splicing

GENE3200 Bedell Chapter 8 Oct 8, 2012

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The central dogma of biologySanders & Bowman Fig. 1.8

GENE3200 Bedell Chapter 8 Oct 8, 2012

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RNA 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)

GENE3200 Bedell Chapter 8 Oct 8, 2012

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Comparison of DNA and RNA

CharacteristicBases Sugar Strands Base-pairing

DNAG, A, T, C Deoxyribose Double-stranded G:C, A:T

RNAG, A, U, C Ribose Single-stranded G:C, A:U, G:U

GENE3200 Bedell Chapter 8 Oct 8, 2012

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Comparison of DNA replication and transcription

CharacteristicEnzyme Primer needed Site of initiation Synthesis direction Site of termination Product

ReplicationDNA polymerase Yes Origin (Ori) 5 3 Chromosome end ds DNA

TranscriptionRNA polymerase No Promoter 5 3 Termination site ss RNA

GENE3200 Bedell Chapter 8 Oct 8, 2012

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RNA classificationMessenger 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)GENE3200 Bedell Chapter 8 Oct 8, 2012 6

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 (eukaryotesonly)Small interfering RNA (siRNA) discussed in Chapter 12

Catalytic activity (eukaryotes only)RibozymesGENE3200 Bedell Chapter 8 Oct 8, 2012 7

The product of transcription is a single-stranded primary transcriptDNA5' 3' RNA-like 3' strand 5' Template strand

GENE3200 Bedell Chapter 8 Oct 8, 2012

Transcription RNA5' 3' Primary transcrip t

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 transcription8

Know how to answer questions like theseFor the DNA sequence: 5-CTAGAT-3 What is the sequence of its complementary DNA strand? 3-GATCTA-5 If this is the RNA-like (coding strand), what is the sequence of its RNA transcript? 5-CUAGAU-3 For the RNA sequence: 5-GUCCA-3 What is the sequence of its template (noncoding) strand of DNA? 3-CAGGT-5 What is the sequence of its RNA-like (coding) strand of DNA? 5-GTCCA-3GENE3200 Bedell Chapter 8 Oct 8, 2012 9

General diagram of a gene

The promoter is a DNA sequence that determines where transcription initiates but isnt 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 +1GENE3200 Bedell Chapter 8 Oct 8, 2012 10

Bacterial RNA polymeraseThe core enzyme cannot bind the promoter or initiate RNA synthesis without the subunitGENE3200 Bedell Chapter 8 Oct 8, 2012

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Bacterial 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-3GENE3200 Bedell Chapter 8 Oct 8, 2012 12

Transcription initiation in prokaryotesRNA polymerase holoenzyme binds to the promoter (-10 and -35 sequences) to form the closed promoter complexGENE3200 Bedell Chapter 8 Oct 8, 2012

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

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Transcription elongation and termination in prokaryotesAfter initiation holoenzyme synthesizes 8 10 nt of RNA, then sigma subunit dissociates from the core enzymeGENE3200 Bedell Chapter 8 Oct 8, 201214

Synthesis by core enzyme occurs until a termination sequence is encountered

Termination of transcription in prokaryotesWhen 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

GENE3200 Bedell Chapter 8 Oct 8, 2012

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Two transcription termination mechanisms in prokaryotesTermination 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

GENE3200 Bedell Chapter 8 Oct 8, 2012

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Transcription termination in prokaryotesEach transcript of a gene terminates at the same site Two kinds of terminators in prokaryotes: Rho-dependent termination Requires Rho () factor RNA polymerase pauses, then 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 genesGENE3200 Bedell Chapter 8 Oct 8, 2012 17

Intrinsic termination in prokaryotes

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GENE3200 Bedell Chapter 8 Oct 8, 2012

Intrinsic termination in prokaryotes (cont)Inverted repeat sequences in mRNA form a stem-loop structure (hairpin) Series of Us 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

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Rho-dependent terminationRho 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

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In eukaryotes, transcription is more complex than in prokaryotesEukaryotic 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 eukaryotesGENE3200 Bedell Chapter 8 Oct 8, 2012 21

Eukaryotic transcription uses multiple RNA polymerasesEukaryotes 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

GENE3200 Bedell Chapter 8 Oct 8, 2012

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RNA polymerase II promoter sequences in eukaryotesThe 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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GENE3200 Bedell Chapter 8 Oct 8, 2012

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 GCrich box are more variableGENE3200 Bedell Chapter 8 Oct 8, 201224

Promoter recognition in eukaryotesRNA 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

GENE3200 Bedell Chapter 8 Oct 8, 2012

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Transcription initiation in eukaryotesTFIID consists of the TATAbinding protein (TBP) and a protein called the TBPassociated 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 complexGENE3200 Bedell Chapter 8 Oct 8, 2012 26

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 mRNAGENE3200 Bedell Chapter 8 Oct 8, 2012

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Enhancers and silencers lead to differential expression of eukaryotic genesPromoters alone are not sufficient to initiate transcription of many eukaryotic genes Enhancer sequences Increase the level of transcription of specific genes A