Term
| Regulation of mRNA ½ life |
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Definition
• Great variability in mRNA ½lives (minutes - >48h)
• Sequences determining ½ lives usu. in 3’ untranslated region
• Short ½ life mRNAs – AU rich sequences (AUUUA)
• When necessary to synthesize large amts of proteins, mRNAs are stabilized, resulting in a rapid increase in mRNA levels and pt production
• AUUUA sequences recognized by specific pt’s that either stabilize or destabilize mRNA – mechanism not understood – however linked to PolyA tail
• Closely linked to translation regulation |
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Term
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Definition
• Fe toxic, detoxified by ferritin, so Fe, immediate synthesis of ferritin
• Ferritin mRNA levels constant
• Fe response element (IRE) in 5’ untranslated region of ferritin mRNA, binds IRE-BP which blocks 40S subunit
• When Fe added, binds to IRE-BP and comes off RNA allowing translation of ferritin mRNA
• IRE also present in 5’ of untranslated region of mRNAs involved with heme synthesis (similar regulatory mechanism)
• IRE – cis-acting; IRE-BP – trans-acting |
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Term
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Definition
• mRNAs that can recruit initiation factors most effectively are ones translated @ highest rate
• general translation control through regulation of eIF4E, the cap binding pt by inhibitors that bind it
• specific mRNA translation controlled by interfering w/ or stimulating the formation of the translation initiation complex (bind in 5’ – block, bind in 3’ end, can block or stimulate)
• most rapid way to regulate gene expression
• also critical when no mRNA present – in early development depend on stored maternal mRNA |
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Term
| Regulation of transcription during development |
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Definition
• Much of cell differentiation driven by expression of key TF’s specific for that cell type
• Ex: muscle cell development triggered by family of similar TF’s that activate muscle-specific genes
• Undifferentiated fibroblasts can be converted to myoblasts in culture by expression of factor MyoD; these will then form myotubes; MyoD acts as heterodimer and 2 forms present when cell growing though redundant (like myf5)
• MyoD inhibitor binds to MyoD to prevent it from binding to E2A, keeping genes silent |
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Term
| Steroid hormones and transcription activation |
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Definition
• Separate receptors inside cell for each steroid
• Steroids enter cell, bind to receptors, the steroid+ receptor complex binds to DNA to activate
• Steroid receptors are trans-acting factors that control expression of a number of genes
• Receptors have 3 domains: DNA-binding, ligand binding, activation domains which binds pt’s needed for transcription – positive control
• Steroid receptor binds as a dimmer to repeats
• Steroid needed to change receptor shape or remove repressors from receptor so it can bind to DNA |
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Term
| cAMP activation of liver genes |
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Definition
• CRE – sequence in front of cAMP genes in liver binds to CRE binding pt; one form of CREB phosphorylated (binds to DNA) and other unmodified
• When cAMP , pt kinase A activated, adds PO4 to CREB to activate it; phosphatase removes it when cAMP
• Another example of positive control |
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Term
| Control of rate of active gene transcription |
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Definition
• Depends on ability of the promoter complex of pt’s to recruit and stabilize the binding of TFIID to the promoter that in turn will recruit RNAPoly II and other factors req’d to initiate transcription
• Assembly of complex regulated by presence/ absence of various factors, modification of factors, or binding of small molecules to factors
• TF’s usu. contain a DNA-binding and separate activation domain so removal of activation domain kills its ability to recruit polymerase, TF’s, etc.
• TF’s in different cells determine activity of genes
• Change in rate often result of change in a factor |
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Term
| Control at chromatin level |
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Definition
• heterochromatin – inactivated genes “packed away”, i.e. genes for lipid metabolism in red blood cell
• inactive chromatin usu heavily methylated at CG doublets and doesn’t have highly acetylated lysines in N-terminal region of histones, also tightly packed b/c of 1 histone H1 per nucleosome (more than in active chromatin)
• methylation inherited as hemimethylation – maintenance methylase recognizes this & restore pattern of parental DNA – permanent gene silencing |
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Term
| Proteins can bind sequences on dsDNA |
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Definition
• transcription factors and repressor proteins recognize specific DNA sequences
• often sequences are inverted repeats and can be read by H bonds, ionic bonds, and methyl recognition (likewise may recognize lack of methyl groups) w/o unwinding strands
• typical binding pt – binds short stretch of DNA; often dimers to bind 2 sequences
• common motifs: zinc fingers – aa loops; helix-loop-helix, @helix to fit into major groove; leucine zippers – pt dimers (scissor shape) |
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Term
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Definition
• 3 lactose metabol genes, controlled by 1 promoter
• genes expressed only when lactose present and glucose is absent (b/c glucose is preferred)
• if lac repressor bound near transcription start site, RNA polymerase blocked from binding – pt acts as negative control element; pt unbinds from DNA if bound by lactose
• if CAP pt bound, helps RNA polymerase bind – positive control element; when cAMP drops, CAP comes off of DNA; cAMP synthesized only when cells hungry for glucose (cAMP=hunger signal) |
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Term
| Positive vs. negative control |
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Definition
• always 2 components – cis-acting element (n acid sequence contain info for control system) and trans-acting element that specifically binds to the sequence
• genes regulated together often have same or similar cis and controlled by same trans
• positive control – trans-acting factor binds to the target and the pt-n acid complex stimulates expression of the gene
• negative control – trans-acting factor binds to target the pt-n acid complex represses expression |
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Term
| Regulation of pt steady state |
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Definition
1. transcription rate of gene
2. processing and transport of the pre-mRNA into mRNA that is present in the cytoplasm
3. degradation rate of the mRNA (#1-3= mRNA metabolism)
4. rate of translation of the mRNA
5. stability of the protein |
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Term
Important things to remember about: DNA replication transcription
translation |
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Definition
DNA replication – leading strand read 3’5’, lagging strand read 5’3’, because DNA made 5’3’, lagging strand has to make Okazaki fragments since it can’ continuously replicate DNA as fast as fork moves
Transcription – direction of transcription goes 5’3’ on non-transcribed/sense/coding strand; goes 3’5’ on transcribed/anti-sense strand; b/c mRNA is made 5’3’
Translation- ribosome reads mRNA 5’3’
***so the sense strand is the strand that specifies what protein is made/order of aa’s!!! (p.23 – textbook) |
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Term
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Definition
• final step in mRNA metabolism
• 1/2life=<30min-24h; regulated and often is a step in regulating pt expression
• degradation initiated while mRNA being translated
1. polyA tail removed by polyA specific nuclease, probably breaking 3’ and 5’ interaction/expose cap
2. cap removed by “decapping” enzyme
3. once both removed, further initiation of mRNA translation inhibited, degradation by non-specific exonucleases from either end
• we think info about ½ life stored @ mRNA 3’ end |
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Term
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Definition
• 2 methionine tRNAs for the AUG codon
• m version used in the middle of pt’s and can only bind at A site
• i version only used for initiation and binds 40S ribosome once it finds initiation codon
• this tRNA interacts w/eIF2 (another translation initiation factor) that helps bring i version to the AUG codon and the 40S subunit
• then 60S binds and ribosome complete and ready to start translocation cycles (w/i version @ P site) |
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Term
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Definition
• all pt’s start with AUG codon – methionine aa
• translation starts at 5’ cap and scans until 1st AUG – determines proper reading frame & start site!
• Pt that binds cap (eiF4e) interacts w/ large pt (eIF4G) which is key pt for initiation
• eiF4G also binds 2 other initiation factors eIF4A (RNA helicase that helps ribosome scan mRNA looking for 1st AUG) & eIF3, which in turn binds 40S subunit
• polyA tail also req’d for initiation b/c bound by PABP that also finds to eIF4G and stabilizes 5’ complex |
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Term
| Peptide bond formation, translocation, termination |
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Definition
• When proper tRNA is bound to ribosome, GTP hydrolyzed (which is bound to the tRNA along with EF1) to lock tRNA in place
• rRNA catalyzes formation of peptide bond (proven in past year, specifically 28SrRNA), transferring growing peptide bond from P site to the aa in the A site; potential antibiotic attack
• empty tRNA on P leaves and chain transferred, catalyzed by elongation factor G (EF-G), more GTP
• specific stop codons – recognized by a pt shaped like tRNA that binds to A; signals polypeptide chain to be transferred from P site to water |
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Term
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Definition
• 20 different tRNA synthetases; use ATP for E; responsible for attaching aa to tRNA
• synthetases recognize appropriate tRNAs by binding to whole tRNA molecule; binds both anticodon loop and CCA end so that correct aa is added – crucial to putting right aa on tRNA!
• mRNA bound by 80S ribosome; each ribosome binds 3 tRNAs at once
• “APE” – aa, polypeptide, exit sites |
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Term
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Definition
tRNA structure • all tRNAs end in CCA sequence and aa bound to 2’ or 3’ OH group @ end of tRNA
• tRNAs unusual – contain modified nt’s (dihydrouridine, pseudouridine) which are present on loops that are used to recognize tRNA synthetases and tRNA binding on ribosome
• anticodon – region that is used to decode mRNA |
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Term
| Genetic code and basic pt synthesis |
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Definition
• 64 possible codons, only 20 aa’s so some redundancy (i.e. several stop codons); so 25% of time single base mutation won’t change aa
• no start codon
• 3 possible reading frames, depending on where 1st codon starts
1. each aa attached to tRNA
2. ribsosome selects proper tRNA based on code in reading frame
3. transfer of aa to polypeptide chain |
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Term
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Definition
• Variable # of tandem repeats (VNTR) – aka. Minisatellite (can be used for DNA fingerprinting), microsatellite
• Single nt polymorphs (SNP) – almost exclusively biallelic; ~90% sequence variants; decipher population histories; track somatic changes (i.e. LOH), predict response to therapy; id genes for complex diseases to determine risk and prevention
• Restriction fragment length polymorphism (RFLP) – often due to SNP |
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Term
| Recombination-generated duplication, deletion, insertion |
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Definition
• Loss of heterozygosity (LOH) – frequent somatic mutation associated with cancer
• Pairing of mutant allele with normal allele yields weird recombinations (i.e. deletions, unbalanced translocations, etc.) |
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Term
Mutation hot-spots
Insertions and deletions |
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Definition
• Mutation hot-spots – frequently CpG doublets
• Insertions and deletions can cause:
o Frameshift (if not multiple of 3)
o Loss or gain of codons (if multiple of 3)
• Larger deletions, inversions, fusions, and duplications can create contiguous gene syndromes
• Insertions of L1 or Alu elements – retrotransposition – important in some cases of hemophilia/cancer
• Insertion can include expansion of trinucleotide repeat sequences |
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Term
Germline vs. somatic mutations
NT Substitutions
Transitions vs. transversions |
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Definition
• Germline – heritable; 2x higher in males
• Somatic – non-heritable; greatly increased with mutator phenotypes (mut’s in repair genes)
• Nt Substitutions – missense (aa sub) or nonsense (premature stop codon)
• Can alter RNA processing mutations or regulatory mutations (i.e. promoters) to alter gene expression
• Transition – i.e. sub one purine for other purine
• Transversion – replace purine w/pyrimidine |
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Term
| Pleiotropy, heterogeneity |
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Definition
• Pleiotropy - Multiple phenotypic effects of single gene/gene pair; used in particular when effects not obviously related; ex: HLA genes in multiple diseases
• Heterogeneity – different biochem pathways lead to same disease |
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Term
| Polymorphisms, rare variants, and mutation |
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Definition
• Polymorphisms - Presence in the same population of 2 or more alternative forms of a DNA sequence
• Rare variants – polymorphic difference @ a freq <1% in general population
• Mutation – heritable changes (@cell level) in DNA sequence; source of new variation; inherited mutations dispersed through a population can become a polymorphism |
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Term
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Definition
• many pre-mRNAS can be spliced = multiple pt’s
1. >1pt produced from 1 mRNA in single cell
2. particular pre-mRNA is spliced differently in one cell type than another
• often results in different polyadenylation site
• recognition (or failure to recognize) particular exons – promotion/inhibition of recognition of exons by particular pt’s in cells or different promoters for same genes in different tissues
• yields greater protein diversity (increases proteome); inner ear – slo pt & STREX exon |
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Term
| Ensuring proper donor/acceptor sites & removal of all introns |
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Definition
• exons small
• genetic disease result from defects in splicing; several examples of mutation in 5’ donor site resulting in deletion of exon rather than inclusion of intron in mutant RNA product
• so, cell recognizes exons rather than introns
• definition of terminal exons by cap and donor site for 5’ exon and acceptor site and polyadenylation signal for 3’ exon |
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Term
| Splicing steps w/ factors |
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Definition
• Acceptor site recognized by U2aF pt- binds polypyrimidine tract and U2; bulging of U2 @ branch pt – A sticks out
• Formation of spliceosome triggered; tri-snRNP and U6 bind while U4 bps with U6 until U4 released from U6
• U5 binds 5’ splice site; U1 released
• U6 + U2 + donor site = cleavage of 5’ splice site=form lariat
• Rearrangement of spliceosome w/ U5 interacting w/ 3’ splice site and U6 catalyzing 2nd splice step
• snRPS disassoc from spliceosome; mRNA transp |
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Term
| Trans-acting factors in Splicing |
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Definition
• HnRNP – package pre-mRNA by binding to it; splicing substrates
• snRNAs – req’d for splicing (U1-U6); in snRNP complexes (4 types – U4-6 together)
• lupus – ab’s against snRNP’s?
• U1 – recognize splice acceptor site• |
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Term
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Definition
1. cleavage right before GU, add P to GU and OH to 3’ of exon
• GU covalently joined to 2’OH of adenosine ~25-40 nt’s from 3’ end of intron lariat intermediate
• adenosine = branch pt; consensus seq. there is UACUAAC
• no delta E = make + break phosphodiester bond
2. bond after AG broken, releasing intron & leaving 2nd exon w/ a 5’ PO4; joining 2 exons (no deltaE)
• intron released as circle with short tail |
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Term
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Definition
• many pre-mRNAS can be spliced = multiple pt’s
1. >1pt produced from 1 mRNA in single cell
2. particular pre-mRNA is spliced differently in one cell type than another
• often results in different polyadenylation site
• recognition (or failure to recognize) particular exons – promotion/inhibition of recognition of exons by particular pt’s in cells or different promoters for same genes in different tissues
• yields greater protein diversity (increases proteome); inner ear – slo pt & STREX exon |
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Term
| Ensuring proper donor/acceptor sites & removal of all introns |
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Definition
• exons small
• genetic disease result from defects in splicing; several examples of mutation in 5’ donor site resulting in deletion of exon rather than inclusion of intron in mutant RNA product
• so, cell recognizes exons rather than introns
• definition of terminal exons by cap and donor site for 5’ exon and acceptor site and polyadenylation signal for 3’ exon |
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Term
| Splicing steps w/ factors |
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Definition
• Acceptor site recognized by U2aF pt- binds polypyrimidine tract and U2; bulging of U2 @ branch pt – A sticks out
• Formation of spliceosome triggered; tri-snRNP and U6 bind while U4 bps with U6 until U4 released from U6
• U5 binds 5’ splice site; U1 released
• U6 + U2 + donor site = cleavage of 5’ splice site=form lariat
• Rearrangement of spliceosome w/ U5 interacting w/ 3’ splice site and U6 catalyzing 2nd splice step
• snRPS disassoc from spliceosome; mRNA transp |
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Term
| Trans-acting factors in Splicing |
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Definition
• HnRNP – package pre-mRNA by binding to it; splicing substrates
• snRNAs – req’d for splicing (U1-U6); in snRNP complexes (4 types – U4-6 together)
• lupus – ab’s against snRNP’s?
• U1 – recognize splice acceptor site• |
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Term
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Definition
1. cleavage right before GU, add P to GU and OH to 3’ of exon
• GU covalently joined to 2’OH of adenosine ~25-40 nt’s from 3’ end of intron lariat intermediate
• adenosine = branch pt; consensus seq. there is UACUAAC
• no delta E = make + break phosphodiester bond
2. bond after AG broken, releasing intron & leaving 2nd exon w/ a 5’ PO4; joining 2 exons (no deltaE)
• intron released as circle with short tail |
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Term
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Definition
• Need to define ends of introns/exons; requires a donor site (5’ of intron) and acceptor site (3’)
• Consensus sequence at donor site – always GU for 1st 2 nt’s of intron; consist of 3 nt’s from exon and 1st 7 nt’s of intron
• Cons seq. at acceptor site – ends in AG
• Intron = GU . . . AG |
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Term
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Definition
• RNAPolyII doesn’t terminate transcription precisely – 3’ end formed by cleavage of transcript to removed from template; addition of Poly(A) coupled to cleavage rxn
• 1) AAUAAA sequence in pre-mRNA near 5’ cleavage site; 2) Gu-rich sequence – near 3’ of cleavage site these are cis- elements
Cleavage occurs after a CA or UA following AAUAAA
CPSF – binds to AAUAAA; CstF to GU rich & interacts w/ CPSF; CFI, CFII binds & cleaves pre-mRNA, release 3’end; polyapolym. Adds tail + dissociates before cytoplasm; PABP in cytoplasm |
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Term
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Definition
• 5’ end – cap; appearance of 3’ end; protects from degradation and is req’d for translation
• all RNAs synth by RNApolyII contain 5’cap
• 3’ end – polyA tail; added postranscriptionally
• 1 mRNA = 1 pt
• 5’ unstranslated region (5’UTR) before 1st AUG
• read in reading frame until stop codon reached
• followed by 3’ UTR, ending in polyA tail
• sequence of mRNA different from DNA!! – introns, esp. in 5’ UTR and coding region, not in 3’ UTR
• pre-mRNA = copy of DNA, then splicing |
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Term
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Definition
1. 28SrRNA = 5000nts, 2 small rRNAs (.5S + 5.8S), and ~50 pt’s
2. 18srRNA = 2000nts, 30 pt’s
• rRNA genes = 400 copies in genome; located in nucleolus where rRNA synthesis and processing occur
• multiple copies of 50kbp tandemly repeated sequence
• cleavage of pre-rRNA precursor to 28S + 5.8S + 18S
• requires snoRNAs |
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Term
| RNA processing (overview) |
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Definition
• Eucaryotes - RNA synthesized in nucleus
• Prior to export to cytoplasm, RNA processed (covalently modified) & packaged w/ pt’s
• New mRNA contains exons (expressed) and introns
• Spliceosome removed introns from mRNA
• polyA tail added to mRNA
• mammalian cells – exons in pre-mRNA id’d by spliceosome
• snRNPS – crictial nuclear ribonucleopt’s in spliceosome – RNA component catalyzes splicing |
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Term
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Definition
• Random – some cells inactivate paternal/maternal; some genes escape inactivation (somatic cells)
• Remembered in subsequent cell divisions
• Females are mosaic
• X-inactivation center at Xq13 – controls initiation and propagation of X-inactivation, XIST gene
• Xist RNA coats chromosome to be inactivated = Barr Body
• Chromatin remodeling pt’s co-localize with Xist RNA, covalent modification of histones
• Blurs distinction between dom/rec x-linked conditions |
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