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  1. 7 RNA Synthesis and Processing • Chapt. 7 RNA synthesis and processing brief • Student learning outcomes: • 1. Explain transcription in Prokaryotes • 2*. Explain how Eukaryotic transcription is complex: RNA Polymerases, General Transcription Factors • 3. Briefly explain complex regulation of transcription in Eukaryotes: many proteins • 4*. Describe RNA processing: Cap, polyA, splicing • 5. Describe RNA turnover, Nonsense-mediated decay

  2. Introduction Regulation of gene expression allows cells: to adapt to environmental changes is responsible for distinct activities of differentiated cell types that make up complex organisms. Transcription is synthesis of primary RNA from gene. first step in gene expression (+1 = start of RNA) initial level at which gene expression is regulated. RNAs in eukaryotic cells are modified and processed Gene structure: promoter, +1, 5’-UTR, coding region, 3’-UTR, terminator

  3. Transcription in Prokaryotes Prokaryotes: E. Coli RNA polymerase (RNAP) polymerizes ribonucleoside 5′-triphosphates (NTPs), 5′ to 3′ directed by DNA template Bacterial RNAP has 5 types of subunits. Sigma (σ)subunit is weakly bound; required to identify correct sites (promoter) for initiation. Different s can direct RNAP to different start sites under different conditions Fig. 7.1 Fig. 7.5 Crab-claw shape Active site channel

  4. Transcription in Prokaryotes Promoter - gene sequence to which RNAP binds to initiate transcription. Prokaryote promoters: 6 conserved nucleotides 10 and 35 bp upstream of transcription start site (+1). Consensus sequences - bases most frequently found in different promoters. Transcription initiates de novo (no primer required) major step for regulation of gene expression Fig. 7.2 prokaryotic promoter

  5. Fig 7.3 DNA footprinting DNA footprinting identifies sites RNAP binds: DNA fragment labeled one end (radioisotope or fluorescent dye) Incubated with RNAP, Partially digest with DNase. Boundprotein protects DNA from DNase digestion, Identify sequences compared to DNA with no protein. Fig. 7.3

  6. Fig 7.4 Transcription by E. coli RNA polymerase Initiation:σbinds specifically to –35 and –10 sequences, starting transcription Initial binding is closed-promoter complex because DNA is not unwound During elongation, RNAP maintains unwound region of 15 base pairs.

  7. Fig 7.6 Transcription termination Termination: RNA synthesis continues until RNAP encounters termination signal. • Common signal is symmetrical inverted repeat of GC-rich sequence followed by seven A residues • Transcription of GC-rich inverted repeat results in segment of RNA that can form stable stem loop structure. • disrupts association with DNA template, terminates transcription Fig. 7.6

  8. Transcription in Prokaryotes Prokaryotic gene regulation: control of transcription by interaction of regulatory proteins with specific DNA sequences Cis-acting control elements only affect expression of linked genes on same DNA molecule (e.g. operator). Trans-acting factors can affect expression of genes located on other chromosomes (e.g. repressor). lac operon is an example of negative control—binding of repressor blocks transcription. lac operon is an example of positive control—binding of cAMP to CAP protein enhances transcription .

  9. Fig 7.8 Negative control of the lac operon lac operon: negative regulation of transcription: Enzymes metabolize lactose Z is b-galactosidase gene; Repressor (i) binds O and blocks transcription; Presence of lactose binds to allosteric site on R, R is inactive; transcription starts Fig. 7.8

  10. Fig 7.9 Positive control of the lac operon by glucose lac operon: Positive control of transcription in E. coli : Presence of glucose (preferred energy source) represses expression of genes for enzymes that break down other sugars, such as lac operon Low glucose activates synthesis of cAMP, binds CAP protein, activates expression of operon (still need lactose inactivate R) Fig. 7.9

  11. 2. Eukaryotic cells have 3 nuclear RNAP transcribe different classes of genes: • Pol I, Pol II, Pol III • Pol II makes mRNA • Each has12 to 17 different subunits. • 9 conserved subunits, • 5 of which are related to subunits of bacterial RNAP

  12. Eukaryotic RNA Polymerases and General Transcription Factors Pol II synthesizes mRNA requires initiation factors not associated with RNAP Structure by Roger Kornberg – Nobel Prize General transcription factors (GTFs) - proteins involved in transcription from pol II promoters 10% of human genes transcription factors (GTF, regulatory) Promoters have different regulatory sequences. Fig. 7.10 pol II

  13. Fig 7.11 Formation of a polymerase II preinitiation complex in vitro • Pol II Promoter sequence elements include TATA box • (resembles –10 sequence element of bacterial promoters) • Minimum of 5 GTFs required forin vitro initiation of transcription. • TFIID (TBP + TAFs), TFIIB, TFIIF, RNAP, TFIIE, TFIIH,

  14. Fig 7.13 RNA polymerase II/Mediator complexes and transcription initiation Additional factors required to initiate transcription: • Mediator, large protein complex of more than 20 subunits • interacts with both GTFs and pol II CTD of pol II is phosphorylated, binds other factors that assist processing, elongation, termination Fig. 7.13

  15. Fig 7.20 Regulation: Action of enhancers • cis-acting sequences regulate expression • Transcription factors bind upstream DNA sequences (enhancers) • Activity of enhancers doesn’t depend on distance, orientation to initiation site. Fig. 7.20 enhancer sequences

  16. Fig 7.21 DNA looping Enhancers, like promoters, bind transcription factors that regulate pol II DNA looping allows transcription factor bound to distant enhancer to interact with proteins associated with pol II /Mediator complex at promoter DNA is bound in chromatin: (nucleosomes wrap DNA) modifications of chromatin structure affect regulation Fig. 7.21

  17. Fig 7.23 Technique: Electrophoretic-mobility shift assay EMSA (Electrophoretic-mobility shift assay) identifiestranscription factor binding sites in vitro Radiolabeled DNA fragments incubated with protein, then electrophoresed through nondenaturing gel. Migration of DNA fragment through gel slowed by bound protein Binding sites are usually short DNA sequences (6–10 base pairs), and are degenerate. Fig. 7.23

  18. Fig 7.25 Technique: Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) identifies DNA regions that bind transcription factors in vivo. • Cross-link TF to their specific DNA sequences • Chromatin is extracted, fragmented. Immunoprecipitation isolates fragments of DNA linked to TF • PCR identifies fragments Fig. 7.25

  19. Fig 7.27 transcriptional activators Transcriptional activators bind to regulatory DNA sequences and stimulate transcription. Factors have two independent domains: • one binds DNA (common motifs), • other stimulates transcription by interacting with other proteins (such as Mediator, pol II, coactivators) Less common, gene expression regulated by repressors - bind DNA sequences, inhibit transcription Fig. 7.27 Fig. 7.31

  20. RNA Processing and Turnover 4. RNA processing and turnover: Primary transcript of gene is only the first step: Most newly-synthesized RNAs must be modified, except bacterial mRNAs which are used immediately for protein synthesis while still being transcribed. rRNAs and tRNAs must be processed in both prokaryotic and eukaryotic cells. Regulation of processing provides another level of control of gene expression.

  21. Fig 7.43 Processing of ribosomal RNAs • rRNAs derive from long pre-rRNA molecule • Pol I synthesizes eukaryotic rRNAs (tandem repeat genes) • 5S rRNA in eukaryotes is transcribed from separate gene. Fig. 7.43

  22. Fig 7.44 Processing of transfer RNAs (Part 1) • tRNAs start as pre-tRNAs, (prokaryotes and eukaryotes) • Processing of 5′ end of pre-tRNAs - cleavage by RNase P. • RNase P is ribozyme: RNA has catalytic activity • Processing of 3′ end of tRNAs: cleavage by nuclease, • addition of CCA terminus, (site of aa attachment) • Some bases modified. Fig. 7.44

  23. RNA Processing and Turnover **Eukaryotepre-mRNAs extensively modified before export from nucleus: 5’-CAP, splicing, 3’-cleavage/polyA addition Transcription and processing are coupled. C-terminal domain (CTD) of RNA pol II plays key role in coordinating processes: After phosphorylation, binds other protein complexes: Capping enzymes bind phosphorylated CTD after initiation Cap added after transcription of first 20 to 30 nucleotides

  24. Fig 7.45 Processing of eukaryotic messenger RNAs 5′ end of transcript modified by addition of 7-methylguanosine cap Fig. 7.45

  25. Fig 7.46 Formation of 3´ ends of eukaryotic mRNAs • At 3′ end, poly-A tail is added by polyadenylation. • Signals for cleavage, polyadenylation include: • conserved hexanucleotide (AAUAAA in mammalian cells), • G-U rich downstream sequence element. • Pol II not recognize termination sequences: • stops after mRNA cleaved. Fig. 7.46

  26. Fig 7.48 Splicing of pre-mRNA • **Splicing: Introns(noncoding) removed from pre-mRNA • Splicing proceeds in 2 steps: (lariat intermediate) • 1. Cleavage at 5′ splice site (SS), joining 5′ end of intron to A within intron (branch point). Intron forms loop. • 2. Cleavage at 3′ splice site, ligation of exons, excision intron • Important sequences: • 5’ splice site • 3’ splice site • Branch point • In vitro experiments • (gel electrophoresis, • mutant sequences) Fig. 7.47

  27. RNA Processing and Turnover Spliceosomes: large complexes that splice: 5 types of small nuclear RNAs (snRNAs)— U1, U2, U4, U5, and U6. 6-10 protein molecules small nuclear ribonucleoprotein particles (snRNPs); RNAs are catalytic Classic expt by Joan Steitz identified snRNPs: antibody from Lupus patients: antigens SM, RNP 32P-cells; IP proteins, analyze RNAs 1: anti-Sm; 2, normal; 3, anti-RNP, 4, anti-RNP X is nonspecific band

  28. Fig 7.49 Assembly of the spliceosome Spliceosome assembly: 1. U1 snRNP binds to 5′ SS (complementary base pairs) 2. U2 snRNP binds to branch point 3. Other snRNPs join complex, form intron loop 4. Maintain association of 5′ and 3′ exons: so can be ligated followed by excision of intron. Fig. 7.49

  29. Fig 7.51 Self-splicing introns • Some RNAs self-splice: catalyze removal of own introns (absence of other protein or RNA factors). • Group I — cleavage at 5′ SS mediated by G cofactor. The 3′ end of free exon reacts with 3′ SS to excise intron. • Group II—cleavage of 5′ SS results from attack by A in intron, results in lariat-like intermediate, which is excised. Fig. 7.51

  30. Fig 7.52 Splicing factors assist spliceosome assembly • Other splicing factors bind specific RNA sequences and recruit U1 and U2 snRNPs to appropriate sites • SR factors bind specific sequences in exons, • recruit U1 snRNP to 5′ SS. • U2AF binds pyrimidine-rich sequences at 3 ′ SS • recruits U2 snRNP to branch point. Fig. 7.52

  31. RNA Processing and Turnover *Alternative splicingoccurs in complex eukaryotes Most pre-mRNAs contain multiple introns: different mRNAs can be produced from same gene. Novel means of controlling gene expression, Increases diversity of proteins that can be encoded Sex determination Drosophila; tissue-specific splicing. Alternative splicing of transformer mRNA regulated by SXL protein, only expressed in female flies. SXL repressor blocks splicing factor U2AF. Fig. 7.53

  32. Alternative splicing • Ex. Dscamgene of Drosophila encodes cell surface protein: connections between neurons in fly brain • 4 sets of alternative exons; • one from each set goes into spliced mRNA. • Exons joined in any combination: alternative splicing potentially yield 38,016 different mRNAs. Fig. 7.54

  33. RNA Processing and Turnover RNA editing: processing (not splicing) that alters protein-coding sequences of some mRNAs. Single base modifications: deamination of C to U, A to I Ex. Editing mRNA for apolipoprotein B, (transports lipids in blood), results in two different proteins: Apo-B100 (4536 aa) synthesized in liver (unedited mRNA) Apo-B48 (2152 aa) synthesized in intestine, edited mRNA (a C changed to U by deamination created stop codon) . Fig. 7.55

  34. RNA Processing and Turnover Aberrant mRNAs can be degraded. Nonsense-mediated mRNA decay eliminates mRNAs that lack complete open-reading frames. Ribosomes encounter premature termination codons, translation stops, defective mRNA degraded Ultimately, RNAs degraded in cytoplasm. Rate of degradation controls gene expression rRNAs and tRNAsvery stable, (both prokaryotes, eukaryotes) high levels of these RNAs (greater than 90% of all RNA) Bacterial mRNAs rapidly degraded: t1/2 2 to 3 minutes. Rapid turnover lets cell respond quickly to changes in environment, such as nutrient availability . .

  35. RNA Processing and Turnover Eukaryotic mRNA half-lives vary; < 30 min to 20 hrs Short-lived mRNAs encode regulatory proteins: levels can vary rapidly in response to environmental stimuli. mRNAs encoding structural proteins or central metabolic enzymes longer half-lives Degradation of mRNAs initiated by shortening poly-A tails. Fig. 7.56

  36. Review questions • How does footprinting identify protein binding sites? • How do enhancers differ from promoters as cis-acting regulatory sites • How are 2 structurally and functionally different forms of apolipoprotein B synthesized in liver and intestine? • 15. What is nonsense-mediated mRNA decay and its significance?