BBMB 405 1st Edition Lecture 36 Outline of Last Lecture XVII. Chapter 31: The control of gene expression in prokaryotesA. IntroductionB. Many DNA-binding proteins recognize specific DNA sequencesC. Prokaryotic DNA-binding proteins bind specifically to regulatory sites in operonsOutline of Current Lecture XVII. Chapter 31: The control of gene expression in prokaryotesD. Gene expression can be controlled at posttranscriptional levelsXVIII. Chapter 32: The control of gene expression in eukaryotesA. Eukaryotic vs Prokaryote transcriptional regulationB. Eukaryotic DNA is organized into chromatinC. The control of gene expression can require chromatin remodelingCurrent LectureXVII. Chapter 31: The control of gene expression in prokaryotesD. Gene expression can be controlled at posttranscriptional levels1. Attenuation: Co-translational control of transcriptiona. Attenuation: turn off gene because metabolite is at high enough concentrations that don’t need be synthesized, example: trpb. High concentrations of Trp present in cell, terminator sequences added and translation stoppedc. Low concentrations of Trp present in cell then ribosome pauses and something prevents stop codon from forming so Trp can be translatedd. Mutually exclusive, share some of the same genese. Terminator and alternate stem loop are mutually exclusiveThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- At high levels of tryptophan: after completion of leader peptide, the third and fourth elements create a stem loop which is a transcription terminator signal- At low levels of tryptophan: when leader peptide is not fully completed the second and third elements create an alternate stem loop and transcription continuesf. Can you guess what types of genes these leaders are from?Many amino acid biosynthesis operons contain attenuatorsa) Threonine operon b) Phenylalanine operon c) Histidine operonXVIII. Chapter 32: The control of gene expression in eukaryotesA. Eukaryotic vs Prokaryotic transcriptional regulation1. Commonalities: DNA-binding proteins that activate or repress genes2. DifferencesEukaryotes ProkaryotesDNA packaged in chromatin DNA relatively accessibleLarge genomes Compact genomesGenerally no operons Genes often in operonsMultiple transcription factors per gene Single factor can control several genesRegulatory DNA sequences often distal from promoterOperator sequences are close to promoterCell type specific regulation (multicellular)Generally nutrient specific regulationB. Eukaryotic DNA is organized into chromatin1. Eukaryotic DNA is organized and compacteda. Nucleosome is fundamental unit of organization- Octomeric (8) histone protein subunits- 147 bp DNA wrapped around- Sites of chemical modification for epigenetic regulationb. Nucleosomes organize into chromatin fiber- Left-handed superhelix- Greatly compacts long strands of DNA- DNA accessibility can regulate transcriptionc. Higher order structures form full chromosomes2. Four types of histones make up nucleosome corea. Highly homologous and adopt very similar 3D conformationsb. Octomer contains (H3)2(H4)2 tetramer and two H2A-H2B dimersc. Histones are highly basic (rich in Arg and Lys)3. Assembly of nucleosome core histones: H3 + H4  H3-H4 dimer + H3-H4 dimer  H3-H4 tetramer; H2A + H2B  H2A-H2B dimer; Two H2A-H2B dimer + two H3-H4 tetramer  Histone Octamer4. Nucleosomes organize DNAa. 147 bp of DNA wraps around histones in left handed solenoid arrangementb. Nucleosomes store negative supercoils: DNA is under-wound, easier to unwind during transcription and replication5. Nucleosomes are packaged into chromatin fibersa. Left-handed superhelixb. Beads on string condense through internuclosome interactions: tetranucleosome are two stacks of nucleosomes connected by straight linker DNAc. Histones H1 and/or H5 are linkers: not part of nucleosome core, located in interior of fiber6. Chromatin structure strongly impacts transcriptiona. DNase I cleavage of chromatinb. Non-transcribed DNA is densely packed and inaccessible to nuclease (and DNA binding proteins)c. Actively transcribed DNA is packaged less densely and can be susceptible to cleavage by nucleased. Chromatin structure must be relaxed to enable gene expression7. Hypersensitive sites to DNA I: high levels of degradation when sites being expressed, reveal what conditions gene is being expressedC. The control of Gene expression can require chromatin remodeling1. Chromatin remodeling dictates transcription activation at specific sites; yeast transcription factor GAL4a. Controls gene involved in galactose metabolismb. DNA recognition sequence 5’-CGG(N)11CCG-3’c. Contains two zinc-finger DNA-binding domains: only contact 5’-CGG-3’ sequences in major groovesd. About 4000 potential binding sites in yeast genome2. Chromatin immunoprecipitation (ChIP) can reveal binding sites for transcription factorsa. ChIP experiments for GAL4 revealed only 10 out of ~4000 potential binding sites were bound by GAL4 in yeast grown in galactose mediab. 99% of sites are blocked, presumably by chromatin structure3. Mechanisms for altering chromatin structurea. Covalent modification of histone N-terminal tails: histone acetyltransferase transfers Acetyl from acetyl-CoA onto N-terminal tails; they no longer has positive charge and don’t bind to DNA backbone as wellb. Movement, restructuring or removal of nucleosomes: chromatin remodeling complex uses remodeling protein and ATP to remove histones4. Histone acetylation alters interactions with DNAa. Loosens interaction between histone and DNAb. Recruits bromodomain (acetyllysine-binding domain) containing proteins5. Chromatin remodeling complexes (or engines)6. Histones can be modified in multiple ways7. Transcription factorsa. Enhance or decrease transcription initiation- General transcription factors promote low levels of transcription- Activators increase initiation from specific sites- Repressors decrease initiation at specific sitesb. Multiple domains- DNA-binding domains: specific recognition of cis-regulatory elements- Activation domains: interact with transcription machinery (often indirectly through bridged interactions) or remodel chromatinc. Multiple eukaryotic transcription factors work together to control transcription- Can exhibit functional redundancy, modular interactions, and synergistic activity- These features lead to combinatorial