Gene Control CH 16

Gene Expression Prokaryotic v. Eukaryotic

• Prok: organisms regulate genes in response to environment

• Euk: cells regulates genes to maintain homeostasis in organism

Regulatory Proteins

bind to DNA, regulate binding of RNA polymerase to promoter

Regulatory proteins function/structure

Bind to specific DNA sequences to control reg.

• Gain access to bases of DNA at major groove + DNA motifs

• Either block transcription by prevent RNA pol from binding or stimulate it by facilitating RNA pol binding to promoter

DNA-binding Motifs

• Helix-turn helix motif: two a-helical segments linked by nonhelical segment 

  • Homeodomain is special call and is critical in euk. Development  

• Zinc finger motif: several forms, use zinc atoms to coordinate DNA binding 

• Leucine Zipper motif: dimerization motif which region in on subunit interacts with similar region on another subunit forming a zipper-like connection 

Prokaryotic Regulation

Transcription initiation is pos or neg controlled

• positive: increases frequency (activators enhance binding of RNA pol to promoter)

• Negative: decreases frequency (repressors bind to operators)

Positive control regulation

Activators enhance binding of RNA pol to promoter

• increases frequency of transcription

Negative Control regulation

Repressors bind to operators (reg. cites on DNA) that prevent/decrease initiation frequency (by preventing RNA Pol)

• decrease frequency of transcription

Effector Molecules (regulation)

Substances that influence activators and repressors. (inducers)

Induction (prok)

enzymes for certain pathways are produced in response to a substrate

• Inducers: bind to repressor proteins→ allows RNA pol to bind now

Repression (prok)

Gene expression decreases despite potential enzyme production.

• Repressor proteins: bind to operator preventing transcription (sometimes need effector molecule to remain bonded)

Lac Operon

Encodes proteins necessary for use of lactose as energy storage (when glucose is short)

• lac Z,Y,A

• lac repressor lac L

Lac Repressor

Protein that inhibits transcription of the lac operon.

Allolactose

Inducer that prevents lac repressor binding.

Negative Regulation of lac operon

• Lac repressor binds to operator to block transcription 

• In the presence of lactose, an inducer molecule (allolactose) binds to repressor protein 

• Repressor can no longer bind to operator 

• Transcription proceeds 

Glucose Repression

Preference for glucose over other sugars in metabolism.

• Catabolic Activator Protein (CAP): an allosteric protein with cAMP as effector 

• Level of cAMP in cells is reduced in presence of glucoses if not stimulation or transcription from CAP-responsive operons takes place 

Inducer Exclusion

Presence of glucose inhibits lactose transport into the cell.

Operons

Gene clusters regulated together in prokaryotes.

Operator

DNA region where repressors bind to block transcription.

trp Operon

Operon encoding tryptophan biosynthesis genes. (important for making proteins) 

• Operon is not expressed when cell contains sufficient amount s of tryptophan 

• Operon is expressed when levels of tryptophan are low 

Trp Repressor

helix-turn-helix protein that bonds to operator site located adjacent to trp promoter 

Negative Regulation of trp operon

• Trp repressor binds to operator to block transcription 

• Binding of repressor to operator requires a co-repressor which is tryptophan (operon is repressed) 

• When tryptophan levels fall, repressor cannot bind to operator (operon id depressed, versus being induced) 

Co-repressor

Substance required for repressor binding to operator. (trp operon)

Eukaryotic Regulation

More complex; major differences from prokaryotes... 

• Euks. have DNA organized into chromatin (complicates protein-DNA interaction) 

• Euks. transcription occurs in nucleus while translation occurs in cytoplasm 

Amount of DNA involved in regulating euks. genes is much larger

Transcription Factor Nomenclature

General transcription factors are named with letter designating following TF, & roman numeral designation of which RNA pol the factor interacts with 

• Ex. TFI-> transcription factor RNA pol I 

• Ex. TFIID-> transcription factor pol II D 

General Transcription Factors

Proteins necessary for transcription, but only do so at the basal level.

• TFIID recognizes TATA box sequences 

• After TFIID binds TFIIE, TFIIF, TFIIA, TFIIB, and TFIIH bind + many transcription-association factors = TAFs 

• This initiation complex can initiate synthesis at a basal level 

Specific Transcription Factors

Factors that enhance transcription in specific conditions, stimulation higher levels

• Each factor consists of DNA-binding domain & separate activating domain that interacts with the transcription apparatus 

• These domains are independent in the protein 

Promoters

DNA sequences where transcription factors bind.

• Mediate binding of RNA polymerase II to promoter 

Enhancers

binding site of specific transcription factors 

• Act over large distances by bending DNA to form loop to position enhancer closer to promoter 

• Activator protein binds to it + mediator= initiates transcription 

Coactivators & mediators

Coactivators and mediators are also required for the function of transcription factors 

• Bind to transcription factors and bind to other parts of the transcription apparatus 

Transcription complex

• Virtually all genes transcribed by RNA pol II need the same suite of general factors to assemble initiation complex 

• General and specific TFs near promoter were Pol II binds + activators connecting enhancers to transcription apparatus

Chromatin Structure (euk)

DNA are wound around histone proteins to form nucleosomes  

• Nucleosomes & histones complicates process of transcription (restricts access of transcription machinery to the DNA) 

• Chromatin structure is selectively modulated to allow transcription, etc.  

Chromatin modifications

• DNA Methylation 

  • High levels of DNA methylation correlate with inactive genes 

  • Allele-specific gene expression seen in genomic imprinting is least partially due to DNA methylation 

• X-chromosome inactivation 

  • Mammalian females inactive one X chrom. As a form of dosage compensation 

(X-inactivation-specific transcript (Xist) coats entire inactive X chrom. Leading to histone modification )

Histone Modification

Four possible histones can be modified 

• Acetylation, methylation, phosphorylation are all possible modifications 

  • In general acetylation is correlative with active sites of transcription 

• histone acetylases (HATs) (Transcription is increased by removing higher-order chromatin structure preventing transcription )

• Histone deacetylases (HDACs) (remove acetyl groups from histones)

Chromatin-Remodeling Complexes

contain enzymes that modify histones & DNA + alter chromatin structures 

• ATP-dependent chromatin remodeling factors (one class of remodeling factors) 

  • Function as molecular motors 

  • Catalyze 4 diff. Changes in DNA/Histone binding 

  • Make DNA more accessible to regulatory proteins 

Posttranscriptional Regulation (euk)

• Small RNAs (miRNA and siRNA) 

• Alternative splicing 

• RNA editing 

• mRNA degradation 

• RNA-induced silencing complex (RISCP): RNA that can affect gene expression by inhibiting translation by degrading mRNAs 

RNA Editing

Editing mature mRNA transcripts can produce an altered mRNA that is not truly encoded in genome 

• In mammals, RNA editing involves chemical modification for a base to change its base-pairing properties

(mRNA for serotonin (5HT) receptor (brain receptor for opiates) is edited at multiple sites to produce 12 diff isoforms of protein)

Translation Repressor Proteins

control initiation of translation by bonding to start of mRNA which prevents ribosomes from binding 

Protein degradation

• Ubiquitin: attached mark on cells, marking them for destruction 

• Proteases: degrade proteins by breaking peptide bonds, converting protein into its amino acids 

• Proteasome: macromolecular machine that degrades proteins marked with ubiquitin 

 

Ubiquitin-Proteasome Pathway

large cylindrical complex that proteins enter at one end and exit at other as amino acids or peptide fragments 

• marked by Ubiquitin

• loaded into proteasome

• degraded by proteases

RNA-induced Silencing Complex (RISC)

complex of protein & RNA that can affect gene expression by inhibiting translation by degrading mRNAs 

• double sided pre-miRNA is cut with Dicer

• strands of miRNA are loaded into RISC protein

• bind to complementary mRNA

• binding prevents transcription of that sequence