genetics 15

2 roles of insulators

*act as a barrier to chromatin remodeling or histone modifying enzymes *also block the effects of enhancers that exert their effects on neighboring genes via chromosome looping (or other mechanisms)

ATP-dependent chromatin remodeling

ATP hydrolysis is used to drive a change in the locations and/or compositions of nucleosomes for both activation and repression of transcription

exon skipping

The failure to incorporate an exon into the mature RNA because of a repressor protein (exon 2 is spliced out)

repressor

a transcription factor that binds to a silencer and prevents transcription

DNA methylation inhibits transcription by...

binding to an activator protein or by recruiting proteins that cause the chromatin to become more compact

polA binding protein

binds to the polA tail to enhance stability. *as an mRNA ages, the polyA tail is shorted by exonucleases *polyA binding protein can only bind to tails longer than 10-30 adenosines

histone acetyltransferases

enzymes that attach an acetyl group (COCH3) to positively charged lysines within on the amino terminal tails *eliminates + charge *acetylation of histones attracts chromatin remodelers that shift or evict nucleosomes aiding in the transcription of genes

mechanism for looping

insulator called the imprinting control region (ICR) is located between H19 and Igf2 genes. An enhancer is next to the H19 gene, a loop is formed to prevent this enhancer from stimulating the Igf2 gene

zinc finger motif

one alpha helix and two Beta sheet structures held together by a Sn2+ metal ion *also recognizes sequences in the major groove

leucine zipper motif

promotes the dimerization of 2 transcription factor proteins. 2 alpha helices (coiled coil) are intertwined via the leucines *helix-loop-helix motifs mediate protein dimerization as well

insulator

segment of DNA that functions as a boundary between 2 genes *protects/insulates a gene from the regulatory effects of a neighboring gene

histone modifying enzymes

travel with RNA pol II during elongation *carry out histone acetylation, H3 methylation, and H2B ubiquitination- modifications which facilitate histone removal ahead of the traveling RNA pol II

CTC-binding factor

(CTCF) binds to both the unmethylated insulator and the differentially methylated region (DMR)- a currently unmethylated site following the Igf2- gene forming a loop. *the loop blocks the enhancer from stimulating the Igf2 gene.

example of histone variants

*H2A.Z: core histone variant that is usually found in nucleosomes that flank the transcriptional start site of promoters. Plays a role in gene transcription. (more easily removed than H2A) *H3.3 core histone variant that facilitates eviction and replacement of nucleosomes ahead and behind RNA pol II. also promotes open chromatin. plays a role in gene activation. found in transcribed regions of genes. less common in silent genes. *common variants in the +1 nucleosome, both accelerate transcription.

results of ATP-dependent chromatin remodeling

1. change in the position of nucleosomes (shift/change in spacing) 2. evict histones from the DNA creating gaps 3. Replacing histones with histone variants

mechanism of CREB protein

1. extracellular signaling molecule enters through plasma membrane receptor 2. activates G protein 3. activates adenylyl cyclase enzyme 4. catalyzes synthesis of cAMP 5. cAMP binds to protein kinase A enzyme 6. enzyme travels into the nucleus and phosphorylates CREB proteins 7. CREB can now activate RNA pol to stimulate transcription

ways methylation can affect transcription

1. prevent or enhance the binding of regulatory transcription factors to the promoter region 2. methyl-CpG-binding proteins bind to methylated sequences

functions of regulatory transcription factor proteins are controlled through:

1. the binding of small effector molecules (ex: steroids) 2. protein-protein interactions (ex: forming homodimers and heterodimers) 3. covalent modifications (ex: attachment of phosphate group)

homodimer

2 identical transcription factor proteins come together *heterodimer is 2 different transcription factors

DNA methyltransferase

An enzyme that catalyzes the methylation of eukaryotic DNA. Attaches a methyl group to the 5 position of the cytosine base *full methylation: methylation of the cytosine in both strands *hemimethylation: only one strand

de novo methylation

the methylation of DNA that was previously unmethylated *uncommon, regulated event *regulated to occur in a specific cell type or stage of development

maintenance methylation

the methylation of hemimethylated DNA following DNA replication, preserving the methylated condition in future cells *does not act on previously unmethylated DNA

cAMP response element-binding protein (CREB protein)

CREB proteins are regulatory transcription factors that are activated by cell-signaling molecules (primary messenger) *cause an increase in cAMP (cyclic adenosine monophosphate) cAMP is a second messenger

gene regulation of posttranslational modifications

feedback inhibition and covalent modifications regulate protein function

DNA translocases

catalytic ATPase subunit in remodeling complexes that is simialr to other motor proteins *move along DNA

behind RNA pol II

histone deacetylase removes the acetyl groups, favoring re-formation of nucleosomes (maintain fidelity)

gene regulation of transcription

regulatory transcription factors activate or inhibit transcription. The arrangements and composition of nucleosomes influence transcription. DNA methylation (usually) inhibits transcription

DNA methylation

typically inhibits transcription of eukaryotic genes by covalently adding a methyl group to bases in DNA (yeast and drosophila typically have little to no methylation) *methylation does not slow down the movement of RNA polymerase *methylation is retained following DNA replication and inherited in daughter cells

control elements (regulatory elements)

DNA sequences in eukaryotes that are "cis acting" elements (analogous to the operator sites in bacterial promoters) and recognized by regulatory transcription factors

formation of the pre-initiation complex

GTFs (general transcription factors) and RNA pol II are able to bind to the core promoter and form the pre-RC

chromatin remodeling complex families

ISWI family, INO80 family, Mi-2-family. SWI/SNF family (SWI mutants in yeast are defective in mating-type SWItching and SNF mutants create Sucrose Non-Fermenting phenotype)

nucleosome free region (NFR)

NFR is found at the beginning and end of many genes. Nucleosomes tend to be precisely positioned near the beginning and end of a gene, but are less regularly distributed elsewhere. *core promoter is found at NFR for active genes *NFT is 150 bp *not by itself sufficient for gene activation of transcription

histone code hypothesis

Proposes that specific combinations of modifications, as well as the order in which they occur, helps to determine chromatin configuration and influence transcription

other mechanisms for regulating mRNA translation

RNA interference, regulating ribosomes, controlling function of translational initiation factors, RNA binding proteins that stabilize (bind to 3' end)

3' untranslated region (3'-UTR)

Sequence of nucleotides at the 3' end of mRNA between the stop codon and the polyA tail; *does not encode the amino acids of a protein but affects both the stability of mRNA and its translation. *contains destabilizing elements like the AU-rich element (ARE)

alternative splicing

Splicing of introns in a pre-mRNA that occurs in different ways, leading to different mRNAs that code for different proteins or protein isoforms. Increases the diversity of proteins. *allows two or more sequences to be derived from a single gene *not common in yeast (unicellular eukaryotes) but about 70% of all human pre-mRNAs are alternatively spliced

transcriptional start site location in yeast vs. animals

Transcription start site in Yeast: is right before the +1 nucleosome region In animals transcription start site about 60 base pairs ahead (upstream) of the +1 nucleosome region +1 nucleosome contains histone variants H2A.Z and H3.3 *(these variants may also be elsewhere)

steroid receptor

a category of regulatory transcription factors that respond to steroid hormones. Example: glucocorticoid receptor *steroid hormones are signaling molecules that are synthesized by endocrine glands and secreted into the bloodstream

TFIID

a general transcription factor that binds to the TATA box and is needed to recruit RNA pol II to the core promoter for transcription *regulatory transcription factors can help/inhibit TFIID to the TATA box or can enhance/repress the function of TFIID that facilitates its ability to bind to RNA pol II

activator

a transcription factor that binds to an enhancer sequence and enhances the rate of transcription

increasing RNA concentration regulates gene expression

accomplished by regulating/increasing the rate of transcription or by increasing RNA stability (influenced by longer half-lives of RNA transcripts or by long polA tails)

up regulation

action of enhancers: stimulate transcription 10 to 1000 fold

down regulation

action of silencers (regulatory elements that inhibit transcription)

binding of activators

activator proteins bind to enhancer sequences in the NFR (which may be close to the transcriptional start site or far away)

chromatin remodeling and histone modification

activator proteins recruit chromatin remodeling complexes like SWI/SNF and histone modifying enzymes like histone acetyltransferase. (nucleosomes may be moved, histones may be evicted. some are covalently modified)

gene regulation of RNA processing

alternative splicing alters exon choices RNA editing alters the base sequences of mRNAs

immunorecipitation

antibodies are added that recognize specific nucleosomes and cause them to aggregate and precipitate out of solution

closed conformation

chromatin is compact and transcription does not occur

open conformation

chromatin is easily accessible to transcription factors and RNA polymerase so transcription will occurr

elongation

during elongation, histones ahead of the open complex are covalently modified by acetylation and evicted or displaced. *behind the open complex, histones are deacetylated and become tightly bound to the DNA

paternally inherited Igf2 gene is transcriptionally activated (mechanism)

during sperm formation, the insulator and DMR are methylated, CTCF cannot bind to insulator, loop cannot form, and enhancer stimulates the Igf2 gene (transcriptionally activated)

AU-rich element (ARE)

example of a destabilizing element that is found in many short-lived mRNA *AREs are recognized by their consensus sequence AUUUA by proteins, which influence whether the mRNA is rapidly degraded

pre-mRNA processing

example of how gene regulation occurs after an RNA is made: splicing, capping, polA tailing, and RNA editing (before it becomes a functional mRNA)

nuclear localization signal (NLS)

exposed on the glucocorticoid receptor when heat shock proteins are released. allow for the formation of a homodimer

nucleosome spacing in eukaryotes

fairly evenly spaced just downstream from the +1 nucleosome (less regular farther downstream) *end of genes have a nucleosome followed by an NFR (important for transcriptional termination)

tissue-specific genes

genes that are highly regulated and only expressed in a particular cell type *expression is silenced by methylation of CpG islands

house keeping genes

genes that encode proteins required in most cells of a multicellular organism *cytosine bases in the CpG islands are unmethylated (housekeeping genes are expressed!)

structure of core histone proteins

globular domain with a flexible, charged amino terminus (amino-terminal tail) *DNA wraps around globular domain *amino terminal tails can be covalently modified (acetylation, methylation, and phosphorylation

glucocorticoid response element (GRE)

glucocorticoid homodimer enters the nucleus and binds to the GRE next to a particular gene --> this activates transcription *function as enhansers

glucocorticoid receptors

glucocorticoid receptors in the cytoplasm are complexed with heat shock proteins (HSP90) --> glucocorticoid hormones in mammals bind to glucocorticoid receptors (HSP90 is released) --> receptors form homodimer and enter the nucleus *influence nutrient metabolism: promote glucose utilization, fat mobilization and protein breakdown *hormones are produced by endocrine glands

RNA interference

introduction of double-stranded RNA into a cell to inhibit gene expression. degrades homologous mRNAs and provides eukaryotes with protection from invasion by types of viruses that prevent movement of TEs

chromatin immunorecipitation sequencing (ChIP-Seq)

method to map the locations of specific nucleosomes (or other proteins) within a genome *determines the relative positions of nucleosomes from the beginning of a gene to the end

methyl-CpG-binding proteins

methyl binding domain recognizes CG sequences and binds to DNA. then recruits other proteins to the region that inhibit transcription ex: recruits histone deacetylase which converts the chromatin to a closed conformation by removing acetyl groups from histone proteins making it difficult for nucleosomes to be removed from DNA

domain

molecular structures of transcription factor proteins that have specific functions *when a domain has a similar structure in many different proteins, its called a motif (which also somtimes promote protein dimerization)

mediator

protein complex that mediates the interaction between RNA pol II and regulatory transcription factors and controls ability of RNA pol II to progress to the elongation stage *ex: activator protein and mediator are brought together by formation of a loop

alpha helix

protein secondary structure that binds and recognizes bases along the major groove of DNA (specifically the recognition helix for helix turn helix and helix loop helix motifs) ^these recognition helixes contain many positively charged amino acids (arginine and lysine) that interact with the negatively charged DNA backbone

alpha tropomyosin

protein that comes from a gene that undergoes alternative splicing. functions in regulation of cell contraction (in thin filaments of smooth muscle cells) *pre mRNA has 14 exons (6 are constitutive exons) *out of the other 8 alternative exons: -exon 2 encodes part of the a-tropomyosin protein that makes it function for smooth muscle cells - exon 3 is suitable for striated muscle cells (does not contain exon 2)

splicing factors

proteins that are involved in the molecular mechanism for the regulation of alternative splicing *splicing factors may act as repressors that inhibit the ability of the spliceosome to recognize a splice site, or they may enhance the ability of the spliceosome to recognize a particular splice site

transcription factors

proteins that influence the ability of RNA polymerase to transcribe a gene 1. general transcription factors (needed for RNA pol binding to core promoter and for a basal level of transcription) 2. regulatory transcription factors (regulate the rate of transcription of target genes)

CpG islands

regions of the genome near the promoters of genes. *C and G are connected via a phosophodiester linkage *usually 1000-2000bp

cAMP response element (CRE)

signaling molecule activates CREB protein which binds to a DNA response element CRE sequence: 5'- TGACGTCA- 3'

gene regulation of translation

small RNAs called miRNAs and siRNAs silence the translation of mRNA phosphorylation of translational initiation factors may regulate translation. Proteins that bind to the 5' end of mRNA regulate translation mRNA stability may be influenced by RNA binding proteins

in genomic imprinting

specific genes are methylated during oogenesis or spermatogenesis but not both. methylation pattern is maintained after fertilization *key way to silence genes in different cell types

Replacing histones with histone variants

standard histones are incorporated into nucleosomes during DNA synthesis in S phase, and later they're replaced by histone variants via chromatin-remodeling complexes *histone variants for H1, H2A, H2B, H3 NOT for H4 *variants have many roles, we focus on gene regulation

gonadocorticoids

steroid hormones (effector molecules) that bind to steroid receptors (regulatory proteins) that influence growth and function of the gonads *ex: estrogen and testosterone

combinatorial control

the combo of many factors determines the expression of a given gene 1. 1+ activator proteins stimulate RNA pol to initiate transcription 2. 1+ repressors inhibit RNA pol from initiating transcription 3. binding of effector molecules, protein protein interactions, and covalent modifications affect function of activators and repressors 4. regulator proteins alter arrangement of nucleosomes near a promoter 5. DNA methylation inhibits trasncription

orientation independent (bidirectional)

the regulatory element can function in the forward or reverse direction

TFIID and mediator

the two proteins that communicate the effects of regulatory transcription factors *for structural genes in eukaryotes, regulatory transcription factors bind to other proteins that directly bind to and influence RNA pol II

SR proteins

type of splicing factor. proteins on processed mRNA that bind to exon and mark them *carboxyl terminal end is rich in serines (S) and arginines (R) *amino terminal end has RNA binding domain

variation of regulatory element location

usually located a few hundred base pairs upstream from the promoter (can be quite distant however or can be downstream or within introns)