A. Structure of RNA B. Major Classes of RNA C. Transcription in Prokaryotes D. Transcription in Eukaryotes E. Post-transcriptional Processing of Eukaryotic mRNA F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an example G. Transcriptional Regulation in Eukaryotes: Steroid Hormones as an Example

1. Uracil instead of Thymine 2. Ribose instead of Deoxyribose 3. Usually single-stranded 4. May have hairpin loops (e.g. loops in tRNA)

1. Messenger RNA  mRNA  Contains information for the amino acid sequences of proteins 2. Transfer RNA  tRNA  Attaches to an amino acid molecule and interfaces with mRNA during translation 3. Ribosomal RNA  rRNA  Structural component of ribosomes

4. Small nuclear RNA  snRNA  Component of small ribonucleoprotein particles  Processing of mRNA 5. Small nucleolar RNA  snoRNA  Processing of rRNA 6. Small cytoplasmic RNAs  Variable functions; many are unknown

7. Micro RNA  miRNA  Inhibits translation of mRNA 8. Small interfering RNA  siRNA  Triggers degradation of other RNA molecules 9. Piwi-interacting RNA  piRNA  Thought to regulate gametogenesis

1. 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” 2. 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

3. Transcription requires nucleoside triphosphates (NTPs; ATP, GTP, CTP, UTP) as raw materials 4. Nascent RNA strand synthesis (elongation) occurs only in the 5’  3’ direction, with new nucleotides added to the 3’ end of the nascent strand 5. Transcription is catalyzed by DNA-directed RNA polymerases

6. The initiation of transcription occurs when RNA polymerase binds to a “promoter region” upstream from the transcriptional start site 7. Promoter regions typically have short stretches of common nucleotide sequences, found in most promoters, called “consensus sequences” 8. 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. Bacterial RNA polymerase consists of a core enzyme and a sigma factor 10. Bacterial RNA polymerase core has 4 or 5 subunits  α 2 ββ ‘ ω  α 2 ββ ‘ is essential; ω is not 11. Sigma factors ( σ ) are global regulatory units. Most bacteria possess several different sigma factors, each of which mediate transcription from several hundred genes …

11. … for example:  In E. coli, during log (exponential) growth, the major sigma factor present is σ 70  During stationary phase, it is σ S  Shifting from σ 70 to σ S activates the transcription of multiple genes linked to survival during stationary phase 12. Transcription begins when the core RNA polymerase attaches to a sigma factor to form a holoenzyme molecule

13. The holoenzyme binds to a promoter, and the dsDNA template begins to unwind 14. A nascent RNA strand is started at +1 on the template 15. After transcription is initiated, the sigma factor often dissociates from the holoenzyme 16. RNA polymerase moves 3’  5’ along the template, synthesizing the nascent RNA 5’  3’

17. Transcription ends (termination) when RNA polymerase reaches a terminator sequence, usually located several bases upstream from where transcription actually stops 18. Some terminators require a termination factor protein called the rho factor (  ); these are rho- dependent. Others are rho-independent. 19. 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

1. Chromatin in eukaryotes is unfolded to permit access to the template DNA during transcription 2. 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 region Usually contains: TATA box – Consensus sequence at -25 to -30 and other core consensus sequences

2. …  Regulatory promoter region Immediately upstream from the core promoter, from about -40 to -150 Consensus sequences include: OCT box GC box CAAT box

3. 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

1. 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. 2. Almost all eukaryotic genes contain introns: noncoding regions that must be removed from the pre-mRNA. The coding regions are called exons.

3. Introns are removed, and the exons are spliced together, by ribonucleoprotein particles called spliceosomes. 4. mRNA contains a “leader sequence” at its 5’ end, before the coding region. The coding region begins with a translational initiation codon (AUG). 5. 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

6. The coding region ends with one or more translational termination codons (stop codons). 7. At the 3’ end is a noncoding trailer region. 8. A 3’ poly-A tail, consisting of 50 – 250 adenosine nucleotides, is added to the 3’ end by a 3’ terminal transferase enzyme.

1. Operon: A group of genes in bacteria that are transcribed and regulated from a single promoter 2. 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

3. 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 -galactoside permease  lac a gene: Encodes β -galactoside transacetylase  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 )

4. 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

5. Transcription of the lac operon is stimulated by conditions of low glucose concentration  When glucose levels are low:  Adenylate cyclase 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

5. …  When glucose levels are high:  Adenylate cyclase activity is lowered, so cAMP levels are low  This means there is much less cAMP/CAP complex  And there is decreased lac transcription 6. So … E. coli will metabolize glucose first, then lactose when the glucose runs out

1. Steroid hormones are secreted by endocrine gland cells and travel through the bloodstream 2. The steroid enters the cytoplasm of target cells and binds to a cytoplasmic steroid receptor protein 3. The steroid receptor/steroid complex enters the nucleus, where it binds to regulatory sites (typically upstream from specific promoters) 4. Transcription from some promoters may be activated (“turned on”) while transcription from other promoters may be inhibited (“turned off”)

5. 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. 6. So … you may have a series of different transcriptional events over a time course with early, middle, and late events