GEN 4600 L2 RNA Transcription Learning Objectives

Central Dogma

• The information stored in DNA is used to make a functional protein or RNA molecule

• Transcription is the copying of one strand of DNA (the template strand) into an RNA molecule (a transcript)

• The RNA sequence matches the coding strand except U replaces T

• DNA is transcribed into RNA by an RNA polymerase. It copies the coding strand of DNA by using the complementary strand (the template or non-coding strand) as a template

• RNA polymerase separates the DNA strands, and allows ribonucleoside triphosphates to base-pair with the template strand

• To produce a protein from an RNA molecule, the RNA sequence is read by the ribosome - this is translation. The RNA in this process is messenger RNA (mRNA)

List the core DNA features of a typical eukaryotic gene and sketch how they are arranged

• Promoter: Begins the process of Transcription (Initiation) with the binding of RNA Polymerase to the region located in the 5’ end of the gene

• Transcription Start Site (TSS): The first base to be transcribed denoted with +1

• Exons: Coding regions of the gene that remains in the mature RNA after splicing
  Introns: Noncoding regions of the gene that’s spliced out

• Untranslated Region (UTRs): Region of the mRNA that’s transcribed from DNA but doesn’t code for amino acids in a protein

• Enhancers: Short region of DNA that binds to activator proteins to increase the transcription and expression of a specific gene

• Terminator: Region of the DNA that signals RNA to cease elongation

Name the four stages at which eukaryotic gene expression is regulated

• Transcription

• Post-Transcription

• Translation

• Post-Translation

Identify a genotype-by-environment (GxE) interaction and distinguish it from an effect that appears regardless

of environment

The Hy5 protein in seedlings control the length of the stem of the plant, but is degraded if low sunlight is available to allow for growth to reach the light better. If the environment had no effect then there would be no difference in the plant length regardless of sunlight amount

Name the protein that recognizes the core promoter and its DNA-binding domain, identify the core

promoter elements and explain why promoter prediction from sequence alone is unreliable

• The TATA Binding Protein (TBP) is the protein that recognizes the core promoter and its DNA-binding domain. It is apart of the TFIID (Transcription Factor II D) complex

• The core promoter elements are the TATA box, TFIIB Recognition Element (BRE), Initiator Element (INR), and Downstream Promoter Element (DPE found downstream of the transcription start)

• Promoter prediction from the sequence alone is unreliable because the promoter motifs are often short and redundant, and many promoters lack these elements all together

Explain how enhancers and locus control regions act over long distances, using β-globin as the worked

example

• Enhancers bind to proteins that help boost gene activity and Locus Control Regions (LCRs) is a combination of enhancers and insulator elements. They work over long distances by creating a loop in the DNA that physically brings a gene promoter to them which helps the LCR build the RNA Polymerase machinery for transcription

• In the case of β-globin the LCR loops to the embryonic gene promoter and activates it during the embryo stage, then releases it and loops with the fetal globin gene during the fetus stage and releases it again and loops with the adult globin gene during the adult stage

Trace a transcript from template strand to mature RNA, correctly identifying the template strand, the coding

strand, and the +1 start site.

Name the three eukaryotic RNA polymerases (and the two extra plant polymerases) and state what each

transcribes

• RNA Polymerase I: Transcribes Ribosomal RNA (rRNA)

• RNA Polymerase II: Transcribes mRNA and small regulatory RNAs

• RNA Polymerase III: Transcribes tRNA, 5S RNA, and sn RNA

• RNA Polymerases I,V (plants only): Transcribes si RNA

Put the RNA Pol II pre-initiation complex in assembly order and give the job of each general transcription factor

• 1. TFIID binds to the TATA box via the TBP and causes local DNA unwinding

• 2. Some other components of TFIID, called TBP- associated factors (TAFs), mediate recognition of other promoter elements like INR and DPE

• TFIIB is recruited. This recognizes the BRE promoter element and binds asymmetrically, helping to determine the transcription direction

• TFIIA binds, and stabilizes the TBP-DNA interactions

• TFIIF and RNA Pol II are recruitedTFIIE and TFIIH bind (TFIIH catalyzes ATP-powered unwinding of DNA)

Distinguish the allosteric and torpedo models of termination, and say what evidence would separate them

• Allosteric model: RNA polymerase II conformation changes when it reaches and transcribes poly(A) tail or the 3’ end, leading to dissociation from DNA

• Torpedo model: Excess RNA is degraded by ribonuclease which then torpedoes RNA polymerase enzyme, causing dissociation from DNA

Classify a regulatory protein by its DNA-binding motif: helix-turn-helix, zinc finger, bZIP, or bHLH

• Helix-Turn-Helix: Two alpha helices connected by a short turning sequence. The second helix acts as the recognition helix that fits into the major groove of DNA

• Zinc Finger Domain: A small structural domain stabilized by one or more zinc ions (Zn^2+) coordinated by regularly spaced cysteine and histidine residues. An alpha helix inserts into the major groove

• Basic Region-Leucine Zipper (bZIP): Features a continuous alpha helix containing a basic DNA-binding region adjacent to a dimerization zone with regularly spaced leucine residues. The leucines interlock like a zipper to form coiled-coil dimers

• Basic Region-Helix-Loop-Helix (bHLH): Contains two alpha helices connected by a flexible loop that allows protein dimerization, paired with a basic region that grips the major groove of DNA