Transcription, RNA Processing, and Transcriptional Regulation

1. Transcription, RNA Processing, and Transcriptional Regulation Structure of RNA Major Classes of RNA Transcription in Prokaryotes Transcription in Eukaryotes Post-transcriptional Processing of Eukaryotic mRNA Transcriptional Regulation in Prokaryotes:the Lac Operon as an example Transcriptional Regulation in Eukaryotes: Steroid Hormones as an Example

2. A. Structure of RNA • Uracil instead of Thymine • Ribose instead of Deoxyribose • Usually single-stranded • May have hairpin loops (e.g. loops in tRNA)

3. B. Major Classes of RNA • Messenger RNA • mRNA • Contains information for the amino acid sequences of proteins • Transfer RNA • tRNA • Attaches to an amino acid molecule and interfaces with mRNA during translation • Ribosomal RNA • rRNA • Structural component of ribosomes

4. B. Major Classes of RNA • Small nuclear RNA • snRNA • Component of small ribonucleoprotein particles • Processing of mRNA • Small nucleolar RNA • snoRNA • Processing of rRNA • Small cytoplasmic RNAs • Variable functions; many are unknown

5. B. Major Classes of RNA • Micro RNA • miRNA • Inhibits translation of mRNA • Small interfering RNA • siRNA • Triggers degradation of other RNA molecules • Piwi-interacting RNA • piRNA • Thought to regulate gametogenesis

6. C. Transcription in Prokaryotes • Requires a double-stranded DNA template • The DNA strands separate, and only one of the strands is used as a template for transcription • “Template strand” and “nontemplate strand” • Direction and numbering conventions • From the 3’  5’ direction on the template strand is called “downstream” • From the 5’ 3’ direction on the template strand is called “upstream” • The nucleotide at the transcriptional start site is designated “+1” and the numbering continues +2, +3, etc. in the downstream direction • The nucleotide immediately upstream from +1 is designated “-1” (there is no 0); numbering continues -1, -2, etc. in the upstream direction

7. C. Transcription in Prokaryotes • Transcription requires nucleoside triphosphates (NTPs; ATP, GTP, CTP, UTP) as raw materials • Nascent RNA strand synthesis (elongation) occurs only in the 5’  3’ direction, with new nucleotides added to the 3’ end of the nascent strand • Transcription is catalyzed by DNA-directed RNA polymerases

8. C. Transcription in Prokaryotes • The initiation of transcription occurs when RNA polymerase binds to a “promoter region” upstream from the transcriptional start site • Promoter regions typically have short stretches of common nucleotide sequences, found in most promoters, called “consensus sequences” • Common prokaryotic (bacterial) consensus sequences include: • -10 consensus sequence: TATAAT box or Pribnow box • -35 consensus sequence: TTGACA • -40 to -60: Upstream element; repetitive A-T pairs

9. C. Transcription in Prokaryotes • Bacterial RNA polymerase consists of a core enzyme and a sigma factor • Bacterial RNA polymerase core has 4 or 5 subunits • α2ββ‘ω • α2ββ‘ is essential; ω is not • Sigma factors (σ) are global regulatory units. Most bacteria possess several different sigma factors, each of which mediate transcription from several hundred genes …

10. C. Transcription in Prokaryotes • … for example: • In E. coli, during log (exponential) growth, the major sigma factor present is σ70 • During stationary phase, it is σS • Shifting from σ70 to σS activates the transcription of multiple genes linked to survival during stationary phase • Transcription begins when the core RNA polymerase attaches to a sigma factor to form a holoenzyme molecule

11. C. Transcription in Prokaryotes • The holoenzyme binds to a promoter, and the dsDNA template begins to unwind • A nascent RNA strand is started at +1 on the template • After transcription is initiated, the sigma factor often dissociates from the holoenzyme • RNA polymerase moves 3’  5’ along the template, synthesizing the nascent RNA 5’  3’

12. C. Transcription in Prokaryotes • Transcription ends (termination) when RNA polymerase reaches a terminator sequence, usually located several bases upstream from where transcription actually stops • Some terminators require a termination factor protein called the rho factor (); these are rho-dependent. Others are rho-independent. • Messenger RNA in bacteria is often polycistronic, which means that it has the code for >1 protein on a single mRNA molecule; mRNA in eukaryotes is almost always monocistronic

13. D. Transcription in Eukaryotes • Chromatin in eukaryotes is unfolded to permit access to the template DNA during transcription • Eukaryotic promoters • Recognized by accessory proteins that recruit different RNA polymerases(I, II, or III) • Consist of a core promoter region and a regulatory promoter region • Core promoter region is immediately upstream from the coding regionUsually contains:TATA box – Consensus sequence at -25 to -30and other core consensus sequences

14. D. Transcription in Eukaryotes • … • Regulatory promoter regionImmediately upstream from the core promoter, from about -40 to -150Consensus sequences include:OCT boxGC boxCAAT box

15. D. Transcription in Eukaryotes • Eukaryotic RNA polymerases • RNA polymerase I: Synthesizes pre-rRNA • RNA polymerase II: Synthesizes pre-mRNA • RNA polymerase III: Synthesizes tRNA, 5S rRNA, and several small nuclear and cytosol RNAs • Also, the different RNA polymerases use different mechanisms for termination

16. E. Post-Transcriptional Processing of Eukaryotic mRNA • In eukaryotes, mRNA is initially transcribed as precursor mRNA (“pre-mRNA”). This is part of a transcript called heterogeneous nuclear RNA (hnRNA); the terms hnRNA and pre-mRNA are sometimes used interchangably. • Almost all eukaryotic genes contain introns: noncoding regions that must be removed from the pre-mRNA. The coding regions are called exons.

17. E. Post-Transcriptional Processing of Eukaryotic mRNA • Introns are removed, and the exons are spliced together, by ribonucleoprotein particles called spliceosomes. • mRNA contains a “leader sequence” at its 5’ end, before the coding region. The coding region begins with a translational initiation codon (AUG). • A methylated guanosine cap is added to the 5’ end of the mRNA by capping enzymes. The cap is attached by a 5’  5’ triphosphate linkage

18. E. Post-Transcriptional Processing of Eukaryotic mRNA • The coding region ends with one or more translational termination codons (stop codons). • At the 3’ end is a noncoding trailer region. • A 3’ poly-A tail, consisting of 50 – 250 adenosine nucleotides, is added to the 3’ end by a 3’ terminal transferase enzyme.

19. F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example • Operon: A group of genes in bacteria that are transcribed and regulated from a single promoter • Constitutive vs. regulated gene expression • Constitutive gene expression: When a gene is always transcribed • Regulated gene expression: When a gene is only transcribed under certain conditions

20. F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example • The lac operon in E. coli consists of: • 3 structural genes (genes that encode mRNA) • lac z gene: Encodes β-galactosidase • lac y gene: Encodes β-galactosidepermease • lac a gene: Encodes β-galactosidetransacetylase • The lac promoter gene: lac p • The lac repressor gene: lac i (constitutively expressed and transcribed from its own promoter, different from lac p) • The lac operator region: lac o (which overlaps lac p and lac z)

21. F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example • The genes of the lac operon are only transcribed in the presence of lactose (or another chemically similar inducer) • In the absence of lactose, the lac repressor protein binds to lac o (lac operator) and blocks RNA polymerase from binding to the promoter (lac p) • In the presence of lactose: • Lactose in the cell is converted to allolactose • Allolactose binds to the lac repressor protein, causing it to causing it to dissociate from the operator so RNA polymerase can reach the promoter

22. F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example • Transcription of the lac operon is stimulated by conditions of low glucose concentration • When glucose levels are low: • Adenylatecyclase activity is high and the concentration of cyclic AMP (cAMP) is high • cAMP binds to the catabolite activator protein (CAP) • The cAMP/CAP complex increases the efficiency of binding of RNA polymerase to the promoter • So there is increased lac transcription

23. F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example • … • When glucose levels are high: • Adenylatecyclase activity is lowered, so cAMP levels are low • This means there is much less cAMP/CAP complex • And there is decreased lac transcription • So … E. coli will metabolize glucose first, then lactose when the glucose runs out

24. G. Transcriptional Regulation in Eukaryotes: Steroid Hormone as an Example • Steroid hormones are secreted by endocrine gland cells and travel through the bloodstream • The steroid enters the cytoplasm of target cells and binds to a cytoplasmic steroid receptor protein • The steroid receptor/steroid complex enters the nucleus, where it binds to regulatory sites (typically upstream from specific promoters) • Transcription from some promoters may be activated (“turned on”) while transcription from other promoters may be inhibited (“turned off”)

25. G. Transcriptional Regulation in Eukaryotes: Steroid Hormone as an Example • Once the genes that have been activated by the steroid receptor/steroid complex (primary response or early genes) have been transcribed and translated, some of the proteins may act to regulate the expression of other genes (secondary response genes), etc. • So … you may have a series of different transcriptional events over a time course with early, middle, and late events