Genetics of Microorganisms Exam 2

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123 Terms

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Gene expression

process by which info from a gene is used to synthesize RNA and polypeptides, and to affect properties of cells and phenotypes

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Gene regulation

  • level of gene expression can vary under different conditions

  • allows response to environmental changes such as nutrient availability, stress, etc

  • produce different cells types in multicellular species

  • facilitates changes during development

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Transcription (gene regulation)

  • Regulatory transcription factors activate or inhibit transcription

  • Arrangement and composition of nucleosomes

  • DNA methylation

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RNA Modification (gene regulation)

Alternative splicing and RNA editing

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Translation (gene regulation)

Proteins regulate translation or mRNA degradation, RNA interference

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Posttranslation (gene regulation)

Feedback inhibition and covalent modifications regulate protein function

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combinatorial control + 5 factors

most euk genes are regulated by many factors

  1. >1 activator/repressor proteins stimulates/inhibits transcription

  2. activators and repressors modulated by binding of small effector molecules, protein-protein interactions, and covalent modifications

  3. Regulatory proteins alter nucleosomes near promoter

  4. DNA methylation inhibits transcription

  5. Heterochromatin formation inhibits transcription

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Transcription factors

  • proteins that influence ability of RNA pol to transcribe a gene

  • General and regulatory transcription factors

  • contain regions called domains

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General transcription factors

  • Required for binding of RNA pol to core promoter and its progression to elongation stage

  • Necessary for basal transcription

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Regulatory transcription factors

  • Serve to regulate rate of transcription of target genes

  • influence ability of RNA pol to begin transcription of a particular gene

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domains

regions in transcription factors that have specific functions such as DNA-binding, serving as a binding site for small effector molecules, and dimerization

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motif

a domain or a portion of a domain in transcription factors that has a very similar structure in many different proteins

<p>a domain or a portion of a domain in transcription factors that has a very similar structure in many different proteins</p>
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control/regulatory elements/sequences

  • regulatory transcription factors recognize cis regulatory elements within enhancers

  • binding of regulatory transcription factors to regulatory elements affects transcription of gene

  • activators and repressors

<ul><li><p>regulatory transcription factors recognize cis regulatory elements within enhancers</p></li><li><p>binding of regulatory transcription factors to regulatory elements affects transcription of gene</p></li><li><p>activators and repressors</p></li></ul><p></p>
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enhancers

  • DNA regions (50-1000 bp in length) that contains >1 regulatory elements

  • binding of an activator to enhancer increases rate of transcription, causing up-regulation which is 10-1,000x

  • binding of a repressor which inhibits transcription is down-regulation

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orientation independent/bidirectional

  • many regulatory elements within enhancers can function in forward or reverse orientation

  • regulatory elements can be found within a few hundred nucleotides upstream of promoter

  • can also be found thousands of nucleotides away, downstream from promoter or even within introns

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3 common ways function of regulatory transcription factors can be modulated

  1. Binding of small effector molecule

  2. Protein-protein interactions

  3. Covalent modification

<ol><li><p>Binding of small effector molecule</p></li><li><p>Protein-protein interactions</p></li><li><p>Covalent modification</p></li></ol><p></p>
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open vs closed conformation in chromatin

  • Chromatin is very dynamic and can alternate between 2 conformations

  • Open conformation: chromatin is accessible to transcription factors, transcription can take place

  • Closed conformation: tightly packed, transcription is difficult or impossible

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3 things that can affect gene transcription

  1. Positioning of nucleosomes at or near promoters

  2. Presence of histone variants

  3. Covalent modification of histones

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ATP-dependent chromatin remodeling

  • dynamic changes in chromatin structure

  • nucleosomes change position in cells during gene expression and inactivation

  • nucleosome positioning changes in promoter region as part of gene activation

  • transcriptional activators orchestrate changes in chromatin structure

  • energy of ATP hydrolysis used to drive change in location and/or composition of nucleosomes

  • makes DNA more/less amenable to transcription

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Chromatin remodeling complexes

change chromatin structure by

  • Change in position of nucleosomes

  • Eviction of histone octamers

  • Change in composition of nucleosomes

<p>change chromatin structure by</p><ul><li><p>Change in position of nucleosomes</p></li><li><p>Eviction of histone octamers</p></li><li><p>Change in composition of nucleosomes</p></li></ul><p></p>
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DNA translocase

  • catalytic ATPase subunit that All remodeling complexes have

  • Eukaryotes have multiple families of chromatin remodelers: SWI/SNF, ISWI, INO80, Mi-2

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histones

  • H1, H2A, H2B, H3 and H4 are moderately repetitive

  • Human genome contains over 70 histone genes, most code standard histones

  • A few of these are histone variants which have genes that accumulated mutations that alters AAs

  • some variants are incorporated into a subset of nucleosomes to create specialized chromatin

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histone code hypothesis

  • >50 enzymes in mammals that selectively modify amino terminal tails of histones

  • Acetylation, methylation and phosphorylation affect level of transcription and influence interactions between nucleosomes

  • occur in patterns that are recognized by proteins

  • pattern of modifications provide binding sites for proteins that specify alterations to be made to chromatin structure

  • proteins bind based on the code and affect transcription

<ul><li><p>&gt;50 enzymes in mammals that selectively modify amino terminal tails of histones</p></li><li><p>Acetylation, methylation and phosphorylation affect level of transcription and influence interactions between nucleosomes</p></li><li><p>occur in patterns that are recognized by proteins</p></li><li><p>pattern of modifications provide binding sites for proteins that specify alterations to be made to chromatin structure</p></li><li><p>proteins bind based on the code and affect transcription</p></li></ul><p></p>
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histone acetyltransferases (HATs)

  • + charged lysines within core histone proteins can be acetylated

  • attachment of acetyl group (—COCH3) disrupts electrostatic attraction between histone protein and - charged DNA backbone

  • Favors open conformation; is highly reversible

<ul><li><p>+ charged lysines within core histone proteins can be acetylated</p></li><li><p>attachment of acetyl group (—COCH3) disrupts electrostatic attraction between histone protein and - charged DNA backbone</p></li><li><p>Favors open conformation; is highly reversible </p></li></ul><p></p>
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Histone deacetylases (HDACs)

remove acetyl groups from acetylated histones , favors tighter contact between histones and the DNA

<p>remove acetyl groups from acetylated histones , favors tighter contact between histones and the DNA</p>
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DNA methylation

  • covalent attachment of methyl groups (-CH3)

  • carried out by DNA methyltransferase

  • common in some euk species, but not all

  • DNA methylation usually inhibits euk gene transcription

  • yeast and Drosophila have little DNA methylation while vertebrates and plants have abundant DNA methylation

  • in mammals, ~ 2 to 7% of the DNA is methylated

<ul><li><p>covalent attachment of methyl groups (-CH3)</p></li><li><p>carried out by DNA methyltransferase</p></li><li><p>common in some euk species, but not all</p></li><li><p>DNA methylation usually inhibits euk gene transcription</p></li><li><p>yeast and Drosophila have little DNA methylation while vertebrates and plants have abundant DNA methylation</p></li><li><p>in mammals, ~ 2 to 7% of the DNA is methylated</p></li></ul><p></p>
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CpG islands

in vertebrates and plants in many genes near promoters; 1,000-2,000 nucleotides long and contain high number of CpG sites

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housekeeping genes

CpG islands are unmethylated, genes tend to be expressed in most cell types

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tissue-specific gene

  • expression of these genes may be silenced by methylation of CpG islands

  • methylation influences binding of transcription factors

  • methyl-CpG-binding proteins recruit factors that lead to compaction of chromatin

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Transcriptional silencing via methylation

  • Methylation inhibits binding of an activator protein

  • Methyl-CpG-binding protein recruits other proteins that change chromatin to closed conformation

<ul><li><p>Methylation inhibits binding of an activator protein</p></li><li><p>Methyl-CpG-binding protein recruits other proteins that change chromatin to closed conformation</p></li></ul><p></p>
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de novo methylation

Specific genes are methylated in gametes from female or male parent, an infrequent and highly regulated process

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maintenance methylation

Pattern of one copy of the gene being methylated and the other is maintained in resulting offspring

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How methylation is passed from mother to daughter cell

  1. DNA, not previously methylated, becomes methylated by de novo methylation

  2. When a fully methylated segment of DNA replicates, newly made daughter strands contain unmethylated cytosines making it hemimethylated

  3. hemimethylated DNA recognized by DNA methyltransferase, which makes it fully methylated

<ol><li><p>DNA, not previously methylated, becomes methylated by de novo methylation</p></li><li><p>When a fully methylated segment of DNA replicates, newly made daughter strands contain unmethylated cytosines making it hemimethylated</p></li><li><p>hemimethylated DNA recognized by DNA methyltransferase, which makes it fully methylated</p></li></ol><p></p>
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Gene activation

series of events that allow a gene to be transcribed to produce an RNA molecule

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Gene activation for euks controlled by regulatory transcription factors

  • >1 regulatory transcription factors (activators) bind to enhancer

  • activators recruit coactivators (chromatin remodeling complexes and histone-modifying enzymes)

  • RNA pol binds to core promoter to form preinitiation complex

  • RNA pol proceeds to elongation phase and makes RNA transcript

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nucleosome-free region (NFR)

  • where transcriptional start site at core promoter is found

  • region of DNA where nucleosomes are not found

  • ~150 bp in length

  • May be required for transcription

  • Not solely capable of gene activation; many genes that contain an NFR are not being actively transcribed

  • found at beginning and end of many genes

  • Nucleosomes tend to be precisely positioned near beginning and end of a gene, but are less regularly distributed elsewhere

<ul><li><p>where transcriptional start site at core promoter is found</p></li><li><p>region of DNA where nucleosomes are not found</p></li><li><p>~150 bp in length</p></li><li><p>May be required for transcription</p></li><li><p>Not solely capable of gene activation; many genes that contain an NFR are not being actively transcribed</p></li><li><p>found at beginning and end of many genes</p></li><li><p>Nucleosomes tend to be precisely positioned near beginning and end of a gene, but are less regularly distributed elsewhere</p></li></ul><p></p>
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transcriptional start site (TSS)

  • 2 nucleosomes, −1 and +1 nucleosomes positioned on each side of NFR at transcriptional start site (TSS)

  • 60 bp farther upstream and within NFR

  • +1 nucleosome contains histone variants H2A.Z and H3.3 which may also be found in −1 nucleosome and in some nucleosomes that immediately follow +1 nucleosome in transcribed region

  • Nucleosomes downstream from +1 nucleosome more evenly spaced near beginning of gene, but their spacing becomes less regular farther downstream

  • ends of many euk genes have well-positioned nucleosome followed by NFR, important for transcriptional termination

<ul><li><p>2 nucleosomes, −1 and +1 nucleosomes positioned on each side of NFR at transcriptional start site (TSS)</p></li><li><p>60 bp farther upstream and within NFR</p></li><li><p>+1 nucleosome contains histone variants H2A.Z and H3.3 which may also be found in −1 nucleosome and in some nucleosomes that immediately follow +1 nucleosome in transcribed region</p></li><li><p>Nucleosomes downstream from +1 nucleosome more evenly spaced near beginning of gene, but their spacing becomes less regular farther downstream</p></li><li><p>ends of many euk genes have well-positioned nucleosome followed by NFR, important for transcriptional termination</p></li></ul><p></p>
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Transcriptional activation

  • involves changes in nucleosome position and composition and modifications to histone

  • Activators, may bind within NFR near core promoter or at distance enhancers, recruit chromatin remodeling complexes and histone-modifying enzymes to promoter

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Binding of activators (1/4 Changes that occur to form a Pre-initiation Complex in transcription)

Activation protein binds directly to DNA of regulatory elements within enhancer sequences. enhancer can be close or far from transcriptional start site

<p>Activation protein binds directly to DNA of regulatory elements within enhancer sequences. enhancer can be close or far from transcriptional start site</p>
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Recruitment of coactivators (2/4 Changes that occur to form a Pre-initiation Complex in transcription)

  • Activators recruit coactivators

  • increase transcription rate but don’t bind directly to DNA

  • can enhance transcription (chromatin remodeling, histone modification, recruitment or stimulation of the preinitiation complex, and stimulation of transcriptional elongation)

<ul><li><p>Activators recruit coactivators</p></li><li><p>increase transcription rate but don’t bind directly to DNA</p></li><li><p>can enhance transcription (chromatin remodeling, histone modification, recruitment or stimulation of the preinitiation complex, and stimulation of transcriptional elongation)</p></li></ul><p></p>
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Chromatin remodeling and histone modification (3/4 Changes that occur to form a Pre-initiation Complex in transcription)

  • activator protein recruits chromatin remodeling complex and histone-modification enzyme

  • nucleosome may be moved, and histone may be evicted or replaced with variants

  • some histones are subjected to covalent modification.

<ul><li><p>activator protein recruits chromatin remodeling complex and histone-modification enzyme</p></li><li><p>nucleosome may be moved, and histone may be evicted or replaced with variants</p></li><li><p>some histones are subjected to covalent modification.</p></li></ul><p></p>
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Formation of preinitiation complex (4/4 Changes that occur to form a Pre-initiation Complex in transcription)

General transcription factors and RNA pol II bind to core promoter and form preinitiation complex.

<p>General transcription factors and RNA pol II bind to core promoter and form preinitiation complex.</p>
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Formation of open complex (1/4 events during elongation in transcription)

  • DNA strands must be separated into open complex so that one can act as template for RNA synthesis

  • TFIIH has subunit that functions as DNA translocase, separating DNA strands to convert the closed complex to an open complex

<ul><li><p>DNA strands must be separated into open complex so that one can act as template for RNA synthesis</p></li><li><p>TFIIH has subunit that functions as DNA translocase, separating DNA strands to convert the closed complex to an open complex</p></li></ul><p></p>
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Promoter escape (2/4 events during elongation in transcription)

  • At core promoter, GTFs and mediator bind to RNA pol II and prevent it from traveling along template strand

  • For elongation to occur, promoter escape must happen (RNA pol II released from this binding)

  • Phosphorylation of CTD allows promoter escape.

  • Transcriptional activators facilitate switch between initiation and elongation stages.

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Proximal promoter pausing (3/4 events during elongation in transcription)

  • regulates euk genes

  • RNA pol II pauses in RNA synthesis while close to transcriptional start site

  • Involves binding of 2 factors; DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF)

  • to release pause, positive transcriptional elongation factor b (P-TEFb) phosphorylates both DSIF and NELF

  • results in release of NELF and causes DSIF to facilitate elongation

  • RNA pol II can now transcribe rest of gene

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Other proposed roles of pausing during elongation in transcription

  • Regulating level of transcription

  • Helping maintain nucleosome-free region by blocking nucleosome assembly over core promoter

  • Providing time for recruitment of factors in RNA modifications

  • Providing time for binding of transcriptional elongation factors that provide stability and facilitate transcription process

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Histone modifications (4/4 events during elongation in transcription)

Histone-modifying enzymes important in histone removal and replacement through histone acetylation, H3 methylation, and H2B ubiquitination

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steroid receptors

  • Regulatory transcription factors that respond to steroid hormones

  • hormone binds to transcription factor to affect gene transcription

  • Steroid hormones produced by endocrine glands and secreted into bloodstream before being taken up by cells that respond to hormone

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Glucocorticoids

influence nutrient metabolism in most cells, promote glucose utilization, fat mobilization and protein breakdown

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GRE (Glucocorticoid Response Elements)

  • found within enhancers

  • located near dozens of different genes, so hormone can activate many genes

  • consists of 2 sequences that are close together

    • 5’-AGRACA-3’

    • 3’-TCYTGT-5’

  • glucocorticoid hormones enter cell and bind to glucocorticoid receptor subunits that dimerize, enter nucleus, bind to GRE, and activate gene transcription

<ul><li><p>found within enhancers</p></li><li><p>located near dozens of different genes, so hormone can activate many genes</p></li><li><p>consists of 2 sequences that are close together</p><ul><li><p>5’-AGRACA-3’ </p></li><li><p>3’-TCYTGT-5’</p></li></ul></li><li><p>glucocorticoid hormones enter cell and bind to glucocorticoid receptor subunits that dimerize, enter nucleus, bind to GRE, and activate gene transcription</p></li></ul><p></p>
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CREB (cAMP response element-binding) protein

  • regulatory transcription factor that responds to cAMP which acts as a second messenger by activating protein kinase A, which phosphorylates CREB, allowing it to bind to coactivator CBP

  • binding of CBP causes transcription of adjacent gene to be greatly increased

  • Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol

  • becomes activated in response to extracellular cell-signaling molecules that cause an increase in cytoplasmic concentration of cAMP

  • binds to 2 adjacent sites with consensus sequence

    • 5’-TGACGTCA-3’

    • 3’-ACTGCAGT-5’

  • a CRE (cAMP response element)

<ul><li><p>regulatory transcription factor that responds to cAMP which acts as a second messenger by activating protein kinase A, which phosphorylates CREB, allowing it to bind to coactivator CBP</p></li><li><p>binding of CBP causes transcription of adjacent gene to be greatly increased</p></li><li><p>Unphosphorylated CREB can bind to DNA, but cannot activate RNA pol</p></li><li><p>becomes activated in response to extracellular cell-signaling molecules that cause an increase in cytoplasmic concentration of cAMP</p></li><li><p>binds to 2 adjacent sites with consensus sequence</p><ul><li><p>5’-TGACGTCA-3’</p></li><li><p>3’-ACTGCAGT-5’</p></li></ul></li><li><p>a CRE (cAMP response element)</p></li></ul><p></p>
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Gene repression

  • any mechanism that inhibits transcription of a gene, resulting in a lower level of RNA synthesis from that gene

  • can be short-term, by directly inhibiting steps needed for gene activation or long-term, as in gene silencing via formation of heterochromatin

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Repressor

  • protein that binds directly to a DNA sequence, such as a regulatory element within an enhancer, and inhibits transcription

  • exert their effects by interacting with other proteins or protein complexes called corepressors

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Transcriptional Regulation in Bacteria, Archaea, and Eukaryotes

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Chromatin immunoprecipitation (ChIP)

  • technique used to study DNA- protein interactions in cells

  • involves crosslinking proteins to associated DNA, fragmenting DNA, immunoprecipitating protein-DNA complex using antibody, and then analyzing purified DNA

  • allows identification of which proteins are bound to specific DNA regions and can provide insights into gene regulation and epigenetic processes

<ul><li><p>technique used to study DNA- protein interactions in cells</p></li><li><p>involves <u>crosslinking</u> proteins to associated DNA, <u>fragmenting</u> DNA,<u> immunoprecipitating</u> protein-DNA complex using antibody, and then <u>analyzing</u> purified DNA</p></li><li><p>allows identification of which proteins are bound to specific DNA regions and can provide insights into gene regulation and epigenetic processes</p></li></ul><p></p>
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Crosslinking (ChIP)

DNA and proteins are crosslinked in live cells using formaldehyde or UV light (or other crosslinkers), stabilizing their interactions

<p>DNA and proteins are crosslinked in live cells using formaldehyde or UV light (or other crosslinkers), stabilizing their interactions</p>
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Chromatin Fragmentation (ChIP)

crosslinked chromatin is fragmented using sonication or enzymatic digestion to create smaller DNA fragments

<p>crosslinked chromatin is fragmented using sonication or enzymatic digestion to create smaller DNA fragments</p>
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Immunoprecipitation (ChIP)

antibody specific for protein of interest is used to capture DNA-protein complex from fragmented chromatin

<p>antibody specific for protein of interest is used to capture DNA-protein complex from fragmented chromatin</p>
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DNA Purification and Analysis (ChIP)

DNA associated with precipitated protein-DNA complex is purified and analyzed, can involve PCR, microarrays, or ChIP-sequencing (ChIP-Seq) to identify specific DNA regions where protein is bound.

<p>DNA associated with precipitated protein-DNA complex is purified and analyzed, can involve PCR, microarrays, or ChIP-sequencing (ChIP-Seq) to identify specific DNA regions where protein is bound.</p>
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ChiP-Seq

  • Maps locations of specific nucleosomes within a genome

  • Allows determination of the location of nucleosomes, histone variants and where covalent modifications of histones occur

  • Chromatin immunoprecipitation combined with DNA sequencing

  • Performed in species where genome has been sequenced

<ul><li><p>Maps locations of specific nucleosomes within a genome </p></li><li><p>Allows determination of the location of nucleosomes, histone variants and where covalent modifications of histones occur</p></li><li><p>Chromatin immunoprecipitation combined with DNA sequencing</p></li><li><p>Performed in species where genome has been sequenced</p></li></ul><p></p>
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ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing)

  • high-throughput sequencing method used to assess genome-wide chromatin accessibility

  • provides insight into regulatory landscape of genome by identifying regions where DNA is accessible for binding by cellular factors

  • uses hyperactive Tn5 transposase to tag accessible chromatin regions, allowing for identification of open chromatin regions and their interplay with other factors.

<ul><li><p>high-throughput sequencing method used to assess genome-wide chromatin accessibility</p></li><li><p>provides insight into regulatory landscape of genome by identifying regions where DNA is accessible for binding by cellular factors</p></li><li><p>uses hyperactive Tn5 transposase to tag accessible chromatin regions, allowing for identification of open chromatin regions and their interplay with other factors.</p></li></ul><p></p>
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4 steps of ATAC-seq

  1. Cell Lysis and Nuclei Preparation: cell nuclei isolation and gentle lysis to maintain chromatin integrity

  2. Tagmentation: Tn5 transposase used to insert sequencing adapters into open chromatin regions, simultaneously tags and fragments DNA

  3. Library Preparation and Sequencing: tagged and fragmented DNA is then purified, amplified, and sequenced using next-generation sequencing

  4. Data Analysis: sequencing reads are aligned to reference genome, and peak calling algorithms used to identify regions of increased chromatin accessibility

<ol><li><p>Cell Lysis and Nuclei Preparation: cell nuclei isolation and gentle lysis to maintain chromatin integrity</p></li><li><p>Tagmentation: Tn5 transposase used to insert sequencing adapters into open chromatin regions, simultaneously tags and fragments DNA</p></li><li><p>Library Preparation and Sequencing: tagged and fragmented DNA is then purified, amplified, and sequenced using next-generation sequencing</p></li><li><p>Data Analysis: sequencing reads are aligned to reference genome, and peak calling algorithms used to identify regions of increased chromatin accessibility</p></li></ol><p></p>
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Epigenetics

  • mechanisms that lead to changes in gene expression that can be passed and are reversible, but do not involve change in sequence of DNA

  • epimutation: heritable change in gene expression that does not alter the sequence of DNA

  • Epigenetic inheritance: epigenetic changes passed from parent to offspring

  • not all epigenetic changes are passed from parent to offspring

<ul><li><p>mechanisms that lead to changes in gene expression that can be passed and are reversible, but do not involve change in sequence of DNA</p></li><li><p>epimutation: heritable change in gene expression that does not alter the sequence of DNA</p></li><li><p>Epigenetic inheritance: epigenetic changes passed from parent to offspring</p></li><li><p>not all epigenetic changes are passed from parent to offspring</p></li></ul><p></p>
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Targeting a gene for epigenetic modification by a transcription factor

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Targeting a gene for epigenetic modification by a noncoding RNA

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cis-epigenetic changes

  • maintained at a specific site

  • affect only one copy of gene but not other copy

  • maintained during cell division

  • in subsequent cell divisions, methylated copy of gene B is always methylated whereas other remains unmethylated

<ul><li><p>maintained at a specific site</p></li><li><p>affect only one copy of gene but not other copy </p></li><li><p>maintained during cell division</p></li><li><p>in subsequent cell divisions, methylated copy of gene B is always methylated whereas other remains unmethylated</p></li></ul><p></p>
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trans-epigenetic changes

maintained by diffusible factors, such as transcription factors, affects both copies of a gene

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Factors that promote epigenetic changes

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general features of eukaryotic chromatin structure

  • Nucleosomes are basic unit

  • 146 bp of DNA wrapped around octamer of histone proteins (H2A, H2B, H3, and H4)

  • Nucleosomes interact in a zigzag manner

  • Loop domains formed from SMC proteins and CTCFs

  • Chromatin composed of DNA, protein, non-coding RNAs

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Euchromatin

  • regions that are not stained during interphase

  • transcriptionally active

  • occupies a central position in nucleus

<ul><li><p>regions that are not stained during interphase</p></li><li><p>transcriptionally active</p></li><li><p>occupies a central position in nucleus </p></li></ul><p></p>
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Heterochromatin

  • regions that are stained throughout cell cycle

  • greater level of compaction

  • localized along periphery of nucleus; attached to nuclear lamina

  • Gene silencing: inhibition of transcription; may limit access of activators or other aspects of transcription

  • Prevention of transposable element movement: genes needed for transposition are silenced

  • Prevention of viral proliferation: genes needed to produce more viruses are silenced

<ul><li><p>regions that are stained throughout cell cycle</p></li><li><p>greater level of compaction</p></li><li><p>localized along periphery of nucleus; attached to nuclear lamina</p></li><li><p>Gene silencing: inhibition of transcription; may limit access of activators or other aspects of transcription</p></li><li><p>Prevention of transposable element movement: genes needed for transposition are silenced</p></li><li><p>Prevention of viral proliferation: genes needed to produce more viruses are silenced</p></li></ul><p></p>
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Constitutive heterochromatin

  • heterochromatic at same location in all cell types

  • chromosomal location: close to centromere or telomere

  • repeat sequences: many short tandemly repeated sequences

  • DNA methylation: highly methylated on cytosines in vertebrates and plants

  • Histone modifications: H3K9me3 common in constitutive heterochromatin in yeast and animals; H3K9me2 in plants

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Facultative heterochromatin

  • locations vary among different cell types

  • allows silencing of genes in a cell specific manner

  • formation is reversible; depends on stage of development or cell type

  • chromosomal location: multiple sites between centromere and telomere

  • repeat sequences: LINE-type repeats

  • DNA methylation: methylation at CpG islands in gene regulatory regions; silences genes

  • histone modifications: H3K9me3 found in facultative heterochromatin; animals have H3K27me

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posttranslational modifications (PTMs)

  • on amino-terminal tails of histone proteins

  • specific proteins bind to particular PTMs in nucleosomes via protein domains

  • reader, writer, eraser, and/or recruitment domains

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writer vs eraser vs recruitment domains

  • writer domains: addition of PTMs

  • eraser domains: remove PTMs

  • recruitment domains: recruit other proteins, such as chromatin remodelers or chromatin-modifying enzymes

<ul><li><p>writer domains: addition of PTMs</p></li><li><p>eraser domains: remove PTMs</p></li><li><p>recruitment domains: recruit other proteins, such as chromatin remodelers or chromatin-modifying enzymes</p></li></ul><p></p>
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Molecular events leading to heterochromatin with Higher Order Structure

  • histone PTMs

  • binding of proteins to nucleosomes

  • chromatin remodeling

  • DNA methylation

  • binding of non-coding RNAs

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higher-order (reproducible in 3D) structural features in heterochromatin

  • has closer, more stable contacts of nucleosomes with each other via HP1 which recognizes H3K9me3 and bridges nucleosomes; makes them more compact

  • forms closer loop domains, SMCs promote loop domain formation, CTCFs form a crosslink that stabilizes loops

  • binds to the nuclear lamina

  • may undergo liquid-liquid phase separation

<ul><li><p>has closer, more stable contacts of nucleosomes with each other via HP1 which recognizes H3K9me3 and bridges nucleosomes; makes them more compact</p></li><li><p>forms closer loop domains, SMCs promote loop domain formation, CTCFs form a crosslink that stabilizes loops</p></li><li><p>binds to the nuclear lamina</p></li><li><p>may undergo liquid-liquid phase separation</p></li></ul><p></p>
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Lamina-associated domains (LADS)

  • chromosomal regions associated with nuclear lamina (NL); fibrous layer of proteins

  • organize chromosomes into chromatin territories

  • involved in gene repression

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Liquid-liquid phase separation (LLPS)

  • formation of liquid-like compartments formed by macromolecules that become concentrated in a given location and come out of solution

  • nucleolus, located inside nucleus, is formed by LLPS

  • Heterochromatin may undergo LLPS

<ul><li><p>formation of liquid-like compartments formed by macromolecules that become concentrated in a given location and come out of solution</p></li><li><p>nucleolus, located inside nucleus, is formed by LLPS</p></li><li><p>Heterochromatin may undergo LLPS</p></li></ul><p></p>
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HP1 protein

forms a dimer that binds to two nucleosomes carrying H3K9me3 modification, holds two nucleosomes in close association

<p>forms a dimer that binds to two nucleosomes carrying H3K9me3 modification, holds two nucleosomes in close association</p>
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3 stages of formation of facultative and constitutive chromatin

  1. Nucleation: short chromosomal site bound by chromatin-modifying enzymes and chromatin-remodeling complexes

  2. Spreading: adjacent euchromatin is turned into heterochromatin

  3. Barrier: in interphase chromosomes, spreading stops when it reaches a barrier

<ol><li><p><strong>Nucleation</strong>: short chromosomal site bound by chromatin-modifying enzymes and chromatin-remodeling complexes</p></li><li><p><strong>Spreading</strong>: adjacent euchromatin is turned into heterochromatin</p></li><li><p><strong>Barrier</strong>: in interphase chromosomes, spreading stops when it reaches a barrier</p></li></ol><p></p>
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Pattern of heterochromatin before and after cell division

  • pattern of constitutive and facultative heterochromatin is same in daughter cells as it was in mother cell

  • Multicellular species: heterochromatin patterns established during embryonic development

  • Constitutive: same in all cell types

  • Facultative: cell specific

<ul><li><p>pattern of constitutive and facultative heterochromatin is same in daughter cells as it was in mother cell</p></li><li><p>Multicellular species: heterochromatin patterns established during embryonic development</p></li><li><p>Constitutive: same in all cell types</p></li><li><p>Facultative: cell specific</p></li></ul><p></p>
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During and following cell division, heterochromatin structure is maintained by

  • DNA methylation: hemimethylated DNA becomes fully methylated via maintenance methylation

  • Histone modifications: histones recruit chromatin-modifying enzymes and chromatin-remodeling complexes to daughter chromatids

  • DNA pol: recruit chromatin-modifying complexes

  • Local chromatin structure: higher-order structure favors reformation of heterochromatin

<ul><li><p><strong>DNA methylation</strong>: hemimethylated DNA becomes fully methylated via maintenance methylation</p></li><li><p><strong>Histone modifications</strong>: histones recruit chromatin-modifying enzymes and chromatin-remodeling complexes to daughter chromatids</p></li><li><p><strong>DNA pol</strong>: recruit chromatin-modifying complexes</p></li><li><p><strong>Local chromatin structure</strong>: higher-order structure favors reformation of heterochromatin</p></li></ul><p></p>
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ICF syndrome

immunodeficiency, centromere instability, and facial anomalies, can be due to a mutation in a DNA methyltransferase gene

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Roberts syndrome

prenatal growth defects, craniofacial abnormalities, limb malformations, mutations in a gene for an acetyltransferase

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Genomic imprinting

  • form of gene regulation in which offspring expresses copy of gene from one parent but not both

  • in mammals, only Igf2 gene inherited from father is expressed

  • methylation inhibits binding of CTC-binding factor, which allows Igf2 gene to be stimulated by a nearly enhancer

  • CTC-binding factor binds to unmethylated gene and inhibits transcription by stabilizing loop

<ul><li><p>form of gene regulation in which offspring expresses copy of gene from one parent but not both</p></li><li><p>in mammals, only Igf2 gene inherited from father is expressed</p></li><li><p>methylation inhibits binding of CTC-binding factor, which allows Igf2 gene to be stimulated by a nearly enhancer</p></li><li><p>CTC-binding factor binds to unmethylated gene and inhibits transcription by stabilizing loop</p></li></ul><p></p>
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Igf2 gene (genomic imprinting)

  • gene is de novo methylated during sperm formation but not during egg formation

  • methylation occurs at two sites: imprinting control region (ICR) and a differentially methylated region (DMR)

<ul><li><p>gene is <em><u>de novo methylated</u></em> during sperm formation but not during egg formation</p></li><li><p>methylation occurs at two sites: imprinting control region (ICR) and a differentially methylated region (DMR) </p></li></ul><p></p>
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X-chromosome inactivation (XCI)

  • occurs during embryogenesis in female mammals

  • X-inactivation center (Xic) found on X chromosome

  • Xic contains two genes, Xist and Tsix, which are transcribed in opposite directions

<ul><li><p>occurs during embryogenesis in female mammals</p></li><li><p>X-inactivation center (Xic) found on X chromosome</p></li><li><p>Xic contains two genes, Xist and Tsix, which are transcribed in opposite directions</p></li></ul><p></p>
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process of X-chromosome inactivation

  • one of X chromsomes (the one that will become inactive Barr body) begins to express Xist gene, process of choosing inactive X chromosome is not well understood

  • Xist RNA binds to XIC and spreads to both ends of X chromosome

  • Xist RNA recruits proteins to X chromosome that make it into a compact Barr body, inactive with regard to gene expression

  • some genes on this chromosomes may be expressed

<ul><li><p>one of X chromsomes (the one that will become inactive Barr body) begins to express Xist gene, process of choosing inactive X chromosome is not well understood</p></li><li><p>Xist RNA binds to XIC and spreads to both ends of X chromosome</p></li><li><p>Xist RNA recruits proteins to X chromosome that make it into a compact Barr body, inactive with regard to gene expression</p></li><li><p>some genes on this chromosomes may be expressed</p></li></ul><p></p>
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Pioneer factors

  • transcription factors that can recognize and bind to DNA sequences exposed on surface of a nucleosome

  • recruit chromatin-remodeling complexes and histone-modifying enzymes that carry out epigenetic changes (histone eviction and covalent modifications)

  • influence ability of other transcription factors to bind to enhancer sequences

  • can decrease level of DNA methylation by binding to CpG islands, blocking access by DNA methyltransferases

  • involved in activation/silencing of some genes

<ul><li><p>transcription factors that can recognize and bind to DNA sequences exposed on surface of a nucleosome</p></li><li><p>recruit chromatin-remodeling complexes and histone-modifying enzymes that carry out epigenetic changes (histone eviction and covalent modifications)</p></li><li><p>influence ability of other transcription factors to bind to enhancer sequences</p></li><li><p>can decrease level of DNA methylation by binding to CpG islands, blocking access by DNA methyltransferases</p></li><li><p>involved in activation/silencing of some genes</p></li></ul><p></p>
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Pioneer factors during embryonic development

  • Play a role in changing chromatin structure, has positive/negative effects on transcription

  • Drive reprogramming of genome during initial steps of development

  • Enable some genes to be activated/repressed

  • Work in conjunction with nonpioneer transcription factors to promote cell differentiation

  • Prime certain genes for later expression

  • Important in differentiated cells in adults

  • Levels of expression vary during different stages of embryonic development and among different cell types

<ul><li><p>Play a role in changing chromatin structure, has positive/negative effects on transcription</p></li><li><p>Drive reprogramming of genome during initial steps of development</p></li><li><p>Enable some genes to be activated/repressed</p></li><li><p>Work in conjunction with nonpioneer transcription factors to promote cell differentiation</p></li><li><p>Prime certain genes for later expression</p></li><li><p>Important in differentiated cells in adults</p></li><li><p>Levels of expression vary during different stages of embryonic development and among different cell types</p></li></ul><p></p>
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Trithorax group (TrxG) vs Polycomb group (PcG)

  • key regulators of epigenetic changes during development that produce specific cell types and tissues

  • Trithorax group (TrxG): involved with gene activation

  • Polycomb group (PcG): involved with gene repression

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polycomb group complex

  • PRC1 and PRC2

  • repression begins w/ binding of PRC2 to polycomb response element (PRE)

  • leads to trimethylation of lysine 27 on histone H3.

<ul><li><p>PRC1 and PRC2</p></li><li><p>repression begins w/ binding of PRC2 to polycomb response element (PRE)</p></li><li><p>leads to trimethylation of lysine 27 on histone H3.</p></li></ul><p></p>
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3 ways PRC1 inhibits transcription

  • Chromatin compaction: PRC1 causes nucleosomes in target gene to form a knot-like structure

  • Covalent modification of histones: PRC1 covalently modifies histone H2A by attaching ubiquitin molecules

  • Direct interaction with transcription factor: PRC1 directly inhibits proteins involved with transcription, like TFIID.

<ul><li><p><strong>Chromatin compaction</strong>: PRC1 causes nucleosomes in target gene to form a knot-like structure</p></li><li><p><strong>Covalent modification of histones</strong>: PRC1 covalently modifies histone H2A by attaching ubiquitin molecules</p></li><li><p><strong>Direct interaction with transcription factor</strong>: PRC1 directly inhibits proteins involved with transcription, like TFIID.</p></li></ul><p></p>
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paramutagenic vs paramutable

  • Interaction between two alleles at single locus where one allele induces a change in other allele

  • Allele that has this capacity is paramutagenic

  • Allele that has been altered is paramutable

  • Example: b1 locus in maize

<ul><li><p>Interaction between two alleles at single locus where one allele induces a change in other allele</p></li><li><p>Allele that has this capacity is <strong>paramutagenic</strong></p></li><li><p>Allele that has been altered is <strong>paramutable</strong></p></li><li><p>Example: b1 locus in maize</p></li></ul><p></p>
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paramutations

  • weaker expression of a paramutagenic allele is due to epigenetic changes that decrease/silence its transcription

  • epigenetic changes transferred to paramutable allele

  • silencing of paramutagenic alleles occurs using short ncRNA molecules

  • multiple tandem repeat sequences located close to the coding sequences of paramutagenic and paramutable alleles and may be used to make siRNAs

  • functional mop1 gene (mediator of paramutation 1 gene) and RNA-dependent RNA pol is required for paramutation

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microscopy and size

knowt flashcard image
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Light Microscopy

knowt flashcard image
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Nuclear magnetic resonance (NMR)

  • nuclei in a strong constant magnetic field disturbed by weak oscillating magnetic field and produce electromagnetic signal with frequency of magnetic field at nucleus

  • MRI (Magnetic Resonance Imaging) in humans and animals

  • used to determine 3D structure of biomolecules such as proteins, nucleic acids, and their complexes

  • provides information about structure, dynamics, and interactions of these molecules at atomic level

  • can be performed on samples in solution, which is closer to physiological conditions of biomolecules.

<ul><li><p>nuclei in a strong constant magnetic field disturbed by weak oscillating magnetic field and produce electromagnetic signal with frequency of magnetic field at nucleus</p></li><li><p>MRI (Magnetic Resonance Imaging) in humans and animals</p></li><li><p>used to determine 3D structure of biomolecules such as proteins, nucleic acids, and their complexes</p></li><li><p>provides information about structure, dynamics, and interactions of these molecules at atomic level</p></li><li><p>can be performed on samples in solution, which is closer to physiological conditions of biomolecules.</p></li></ul><p></p>
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X-ray crystallography

  • determine 3D structure of biomolecules by analyzing diffraction pattern of X-rays passing through a crystal of molecule

  • provides atomic-level detail, crucial for understanding biological processes and designing drugs

  • diffraction pattern is analyzed to determine e- density distribution within crystal

  • e- density map reveals positions of atoms and allows for construction of 3D model of molecule

  • high resolution, ability to study small/large structures, ability to reveal fine atomic details of well-ordered macromolecules

  • requirement for high-quality crystals, static view of molecules, not in solution structure (crystal contacts)

<ul><li><p>determine 3D structure of biomolecules by analyzing diffraction pattern of X-rays passing through a crystal of molecule</p></li><li><p>provides atomic-level detail, crucial for understanding biological processes and designing drugs</p></li><li><p>diffraction pattern is analyzed to determine e- density distribution within crystal</p></li><li><p>e- density map reveals positions of atoms and allows for construction of 3D model of molecule</p></li><li><p>high resolution, ability to study small/large structures, ability to reveal fine atomic details of well-ordered macromolecules</p></li><li><p>requirement for high-quality crystals, static view of molecules, not in solution structure (crystal contacts)</p></li></ul><p></p>