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 molecules
An electron micrograph of splicesosomes engaged in intron splicing

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The central dogma of biology

Sanders & Bowman Fig. 1.8

GENE3200 Bedell Chapter 8 Oct 8, 2012

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)

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Comparison of DNA and RNA

Characteristic
Bases Sugar Strands Base-pairing

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

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

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Comparison of DNA replication and transcription

Characteristic
Enzyme Primer needed Site of initiation Synthesis direction Site of termination Product

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

Transcription
RNA polymerase No Promoter 5 3 Termination site ss RNA

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RNA 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)
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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
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The product of transcription is a single-stranded primary transcript

DNA
5' 3' RNA-like 3' strand 5' Template strand

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Transcription RNA
5' 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 transcription
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Know how to answer questions like these

For 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-3
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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 +1
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Bacterial RNA polymerase

The core enzyme cannot bind the promoter or initiate RNA synthesis without the subunit
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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-3
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Transcription initiation in prokaryotes

RNA polymerase holoenzyme binds to the promoter (-10 and -35 sequences) to form the closed promoter complex
GENE3200 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 prokaryotes

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

Termination 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

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Two transcription termination mechanisms in prokaryotes

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

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Transcription termination in prokaryotes

Each 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 genes
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Intrinsic termination in prokaryotes

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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 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

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In eukaryotes, transcription is more complex than 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
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Eukaryotic transcription uses multiple RNA polymerases

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

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RNA polymerase II promoter sequences in eukaryotes

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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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 variable
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Promoter 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

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Transcription initiation in eukaryotes

TFIID 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 complex
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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
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Enhancers and silencers lead to differential expression 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

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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 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
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Silencer 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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