BISC 320 Chapter 9 REGULATION OF TRANSCRIPTION

activators

• regulatory proteins that increase trancription

• in bacteria, usually bind upstream of promoter

repressors

• regulatory proteins that decrease transcription

• in bacteria, usually bind overlapping with promoter

DNA-binding motifs

• structural patterns in proteins that allow them to bind specific DNA sequences

• transcriptional regulators contain DNA binding motifs

  • must be able to recognize specific DNA sequences

• helix-turn-helix

  • one helix fits into major groove, making sequence specific contacts with DNA bases

  • often found as a dimer, with the recognition helices spaced to fit in neighboring DNA grooves

• zinc finger

  • contains a central zinc ion coordinated by two cysteines and two histidines

  • proteins often have several zinc fingers, each of which inserts its alpha helix into the major groove (very modular)

• other motifs can mediate DNA binding

• coiled coils → two alpha-helices wind around each other

  • basic region-leucine zipper (bZIP) → made up of two long alpha helix monomers, N terminal ends splay out and sit in the DNA major groove

  • basic region-helix-loop-helix (bHLH) → each monomer consists of two helices joined by a loop, monomers interact with DNA and one another similarly to the bZIP

Regulatory sequences

• DNA regions that control when and how much a gene is transcribed

• bacterial

  • regulatory proteins bind to sequences upstream of promoter

  • sequences recognized by operator sites

    • activators bind immediately upstream of promoter

    • repressors often bind overlapping promoter

    • regulatory sequences can be several hundred bp away, architectural DNA binding proteins can loop DNA

operator

• A DNA sequence where repressors bind to control an operon

architectural DNA-binding proteins

• egulatory sequences can be several hundred bp away, architectural DNA binding proteins can loop DNA so that the loop operator is closer to the promoter

allosteric effectors

• small molecules that can bind directly to the regulatory proteins and change their confirmation

  • confirmation change can alter DNA binding activity or interaction with other proteins

    • i.e. estrogen, tryptophan

operon

• a cluster of genes that are regulated and transcribed together, usually involved in the same process

  • trp and lac operon

• trp repressor (helix-turn-helix protein) does not bind trp operator when tryptophan levels are low → the genes in the trp operon are transcribed, to make tryptophan

  • allosteric effector of the trp repressor

    • without tryptophan (not binded), trp is created

    • with tryptophan (binded), trp isnt created

• CAP activates more than 100 e. coli genes by enhancing RNA pol affinity to promoters

  • low glucose leads to increase in cAMP levels (induces transcriptional response)→ cAMP binds to CAP and increases its binding to DNA (allosteric effector)

  • high glucose, cAMP is low and CAP doesn’t bind DNA → the CAP activated genes are transcribed weakly

• lac operon → metabolizes lactose for energy source

  • low glucose and present lactose, lac operon is expressed to metabolize lactose

    • CAP binds when glucose is low to increase RNA pol affinity for promoter

    • lac repressor binds in the absence of lactose to prevent transcription

      • allolactose → allosteric regulator for the lac repressor

promoter spacing

• RNA pol is weak if spacing between -10 and -35 is not ideal

  • MerR protein can bind to and bend the DNA to effectively change the spacing

  • also can twist the DNA helix to readjust the orientation of the promoter elements

  • allowed RNA pol to bind more efficiently

sensor kinase

• detects the amount of a specific molecule in the environment by becoming autophosphorylated

• cells respond transcriptionally to stimuli outside the cell by utilizing transmembrane proteins

  • sensor domain (outside)

  • membrane

  • kinase domain (inside) → phosphorylated and phosphate is passed to response regulator which can act as transcriptional activator or repressor

response regulator

• protein which acts as a transcriptional activator or repressor

  • regulatory domain is phosphorylated and is connected to effector domain

• a part of signal transduction pathway

attenuation

• the regulation of transcription by competition between ribosome binding and formation of stem-loop structures in the transcript

  • (BACTERIA) dependent of transcription and translation occuring concurrently

  • used to regulate genes needed for amino acid biosynthesis

• trp mRNA has leader sequence containing open reading frame with trp codons

  • low trp → ribosome stalls blocking region 1, stem-loop forms between 2 and 3 (anti terminator)

  • high trp → ribosome proceeds, allowing attenuator to form between 3 and 4

attenuator

• intrinsic terminator formed to allow ribosome to proceed

riboswitch

• RNA segment of an mRNA that bind a small molecule, resulting in a change in the production of protein encoded by that mRNA

  • changes RNA confirmation

• has two regions

  • aptamer

  • expression platform

• can affect protein production by controlling transcription termination, ribosome binding

• adenine riboswitch binds to adenine to regulate transcriptional termination of genes required for adenine synthesis and transport

  • low adenine → anti terminator and transcription proceeds

  • high adenine → adenine binds aptamer to form temrinator

aptamer

• RNA region that binds to small molecule

expression platform

• RNA region which controls the output (i.e. transcription)

enhancers

• regulatory sequences for eukaryotic genes (like the operator for bacteria)

  • often bind multiple regulatory proteins

  • can be several thousand base pairs away

  • can be upstream or downstream of gene

• distal sequences regulate gene expression by looping

co-activators

• proteins that increase transcription without directly binding DNA

  • go to enhancer

• DNA binding proteins do not regulate transcription directly → recruit additional proteins

• DNA binding domain recognizes specific DNA sequence

• additional domains can aid dimerization or interact with additional regulators

co-repressors

• proteins that reduce transcription without directly binding DNA

  • go to enhancer

hyperacetylated

• hyperacetylated chromatin tends to be actively transcribed

• gene activation is often coupled to recruitment of histone acetyltransferases

Regulation of galactose metabolism

• coactivators and allosteric regulation

• recruitment of mediator can promote transcription → coactivator

• Gal4 (DNA binding protein) binds upstream of genes required for galactose metabolism

  • regulated by Gal 80 which can bind to Gal4 and block recruitment of co-activators

• Galactose is an allosteric regulator of Gal3, which when bound to galactose, can sequester Gal80 to cytoplasm

  • Gal4 can tehn recruit Mediator and SAGA (co-activators)

hypoacetylated

• hypoacetylated chromatin has low levels of transcription

• histone deacetylases can repress transcription

Eukaryotic transcriptional regulation via histone modification

• most transcriptional regulators in eukaryotes are activators

• Ume6 can activate and repress transcription based on nutritional cues

  • enough nitrogen and glucose, Ume6 binds DNA and recruits histone deacetylase and nucleosome remodeler → co-repressors (hypoacetylated)

  • absence of nitrogen and glucose, Ume6 is phosphorylated and histone acetyltransferase (co-activator) is recruited

Eukaryotic transcriptional regulation at elongation

• transcription pauses 30-50 bp downstream of start site → elongation can be regulated by restarting paused polymerases

• initiation of transcription occurs efficiently at heat shock response factor genes because nucleosome remodelers keep promoter free of nucleosomes

• transcription stalls (proximal pausing) as the RNA pol complex is not phosphorylated enough]

• Hsf undergoes a confimrational change and binds to Hses

  • hsf intracts with mediator and a kinase which phosphorylates the CTD and allows transcription to resume

Eukaryotic transcriptional regulation by extracellular signals

• nuclear receptor proteins are ligand-regulated transcription factors and have a DNA binding domain and a ligand binding domain

  • i.e estrogen

• ligand binding induces conformational change in nuclear receptor

  • recruit co-activator or repressor

  • enter nucleus if unbound state localizes to cytoplasm

• signal cascades allow a cell to respond to the environment by binding to a receptor protein on the cellular membrane

• signal amplification → phosphorylation of a series of intermediate molecules can transduce the signal from the receptor protein to a transcription factor