Genetics of Microorganisms Exam 2

Gene expression

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

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

Transcription (gene regulation)

• Regulatory transcription factors activate or inhibit transcription

• Arrangement and composition of nucleosomes

• DNA methylation

RNA Modification (gene regulation)

Alternative splicing and RNA editing

Translation (gene regulation)

Proteins regulate translation or mRNA degradation, RNA interference

Posttranslation (gene regulation)

Feedback inhibition and covalent modifications regulate protein function

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

Transcription factors

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

• General and regulatory transcription factors

• contain regions called domains

General transcription factors

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

• Necessary for basal transcription

Regulatory transcription factors

• Serve to regulate rate of transcription of target genes

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

domains

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

motif

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

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

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

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

3 common ways function of regulatory transcription factors can be modulated

1. Binding of small effector molecule

2. Protein-protein interactions

3. Covalent modification

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

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

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

Chromatin remodeling complexes

change chromatin structure by

• Change in position of nucleosomes

• Eviction of histone octamers

• Change in composition of nucleosomes

DNA translocase

• catalytic ATPase subunit that All remodeling complexes have

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

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

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

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

Histone deacetylases (HDACs)

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

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

CpG islands

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

housekeeping genes

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

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

Transcriptional silencing via methylation

• Methylation inhibits binding of an activator protein

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

de novo methylation

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

maintenance methylation

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

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

Gene activation

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

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

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

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

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

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

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)

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.

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.

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 closed complex to open complex

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.

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

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

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

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

Glucocorticoids

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

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

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)

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

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

Transcriptional Regulation in Bacteria, Archaea, and Eukaryotes

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

Crosslinking (ChIP)

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

Chromatin Fragmentation (ChIP)

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

Immunoprecipitation (ChIP)

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

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.

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

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.

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

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

Targeting a gene for epigenetic modification by a transcription factor

Targeting a gene for epigenetic modification by a noncoding RNA

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

trans-epigenetic changes

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

Factors that promote epigenetic changes

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

Euchromatin

• regions that are not stained during interphase

• transcriptionally active

• occupies a central position in nucleus

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

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

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

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

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

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

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

Lamina-associated domains (LADS)

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

• organize chromosomes into chromatin territories

• involved in gene repression

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

HP1 protein

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

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

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

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

ICF syndrome

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

Roberts syndrome

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

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

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)

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

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

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

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

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

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.

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.

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

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

microscopy and size

Light Microscopy

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.

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)