Chapter 6
Blast program for sequence comparisons and blast p-values- test whether 2 or more sequences (protein or DNA) share a common evolutionary origin (p >10^-3 = due to chance)
Lack of relationship between number of genes in a genome and its biological complexity
10-nm versus 30-nm chromatin fibers – condensed chromatin= 30nm wide, “beads-on-a-string” =10nm wide nucleosome core histone composition (2 each of H2A, H2B, H3, and H4) – Histones exists as octamers. Core is wrapped by 147 bp, about 2turns of DNA= CONSERVED IN ALL EUKARYOTES two turns of DNA around histone core (147 bp) variable size of DNA between nucleosomes (15-90 bp) – depends on species structure of 30 nm fiber and role of H1 histone – resting chromatin will be 30nm wide, H1 binds where DNA enters and exits nucleosome core histone tail modifications (acetylation, methylation, phosphorylation) – methylation & DEacetylation  condensing of chromatin (30nm) acetylation  DE-condensing of chromatin (10nm) phosphorylation & ubiquitination  chromatin remodeling euchromatin versus heterochromatin chromosome scaffold – hold the 30nm chromatin loops attached, genes far apart on the chromosome are close at the base of the loops called SARS (Scaffold Associated Proteins) width of fully condensed metaphase chromosomes (500-750 nm) – 500-750nm wide chromosome banding and FISH (fluorescence in situ hybridization) – identification of karyotypes (chromosome composition) allows painting of each chromosome and easily shows CHROMOSOME ABNORMALITIES commeTRANSLOCATIONS
ARS (autonomously replicating sequences) – 100bp sequences scattered throughout the genome, ARS= replication origins
- circular plasmids containing ARS will replicate but segregation = faulty
CEN (centromere) sequences and their general composition in yeast – CEN DNA sequence is SIMILAR for all chromosomes of the same organisms, different b/w organisms/
- CEN associates with >30 proteins that mediate attachment of microtubules to spindle apparatus
- Kinetochore assembles at centromeres and associates with spindle fibers
Telomere sequences and their composition – located at the ends of chromosomes and contain simple G-rich repeat sequences
- 3 ‘ end of G-rich strand extends 12-16 nt beyond 5’ end of complementary C-rich strands and is bound by proteins that protect from exonucleases
- Telomerase (reverse transcriptase) adds telomere sequences to the ends of chromosomes **** carries its’ own internal RNA template **** Chapter 7
Role of bacterial promoter regions and control elements – promoters usually >50 bp away from start site
- Coordinately regulated genes = genes within one operon, only one promoter sequence
- Most E. coli genes have 2 promoters @ -35 and -10
- RNA Pol will span -50  +20
Bacterial repressors and activators
-35 and –10 promoter sequences in prokaryotes
Bacterial RNA polymerase composition
E. coli sigma factors (sigma70 and alternative ones)
– recognize and bind to promoters @ -35 and -10
- Promoters must associate with sigma factors before RNA Pol can bind
- Most genes require sigma 70 (uses TTGACA and TATAAT)
- E. coli has 7 sigma factors
- Sigma 54= encodes genes for nitrogen metabolism
Lactose operon
- Lac operon encodes 3 proteins that metabolize lactose
- Regulation by 3 DNA elements- CAP, PROMOTER, AND OPERATOR o CAP SITE- when lactose is present and glucose levels are ↓ cAMP is synthesized and binds to adimer of Catabolite Activator Protein
 This cAMP-CAP complex binds just upstream of lac promoter causing increased transcription
- When lactose is present it binds to the repressor causing a conformational change which dissociates from operator - (+) lactose (-) glucose = cAMP is synthesized and binds with CAP which promotes transcription
- (+) lactose (+) glucose = lactose binds repressor allowing sigma70 to read promoter, but little transcription
Coordinate regulation- multiple genes in an operon that utilize one promoter cAMP (cyclic AMP) – 3’ -5’ cyclic- adenosine monophosphate
CAP (catabolite activator protein) – dimeric protein that binds cAMP to increase transcription rate of lacz
Glutamate synthetase gene (glnA) and nitrogen regulatory protein C (NtrC)
- Sigma 54 binds to glnA promoter and is activated by NtrC to start transcription
- NtrC binds to an enhancer region 80-160 bp upstream of start site o Forms a DNA loop o A protein kinase phosphorylates NtrC which then hydrolyzes ATP for activation of sigma 54
Two-component systems in bacteria (sensor protein, response regulator protein, histidine kinase) – binding of a nutrient to the sensor protein activates a Histidine kinase activity which activates a response regulator protein
- Sensor protein DOES NOT bind promoters
- Response regulator proteins DO bind promoters
Bacterial tryp operon and regulation of elongation
- (+) tryptophan available, tryp repressor reduces transcription of operon
- Attenuation occurs where elongation is prohibited o “Attenuator sequence” (4 domains) exists b/w the trp leader RNA (first 140nt) and the gene sequence o Hi level of tryptophan allows ribosomes to translate domains 1 & 2 and forms a hairpin structure at domains 3 & 4  termination of tryp transcription o Lo level of try cause ribosome to stall at domain 1  different hairpin structure @ domains 3 & 4 that DO NOT terminate transcription
Eukaryotic transcription factors and control elements
- Pol II TF’s are all MULTIMERIC and include TFIIA, TFIIB,TFIID o TFIID consists of a single TBP and 13 TAF’s (TBP-associate factors) o Only the TBP subunit of TFIID is required to initiate assembly @ TATA boxes in vitro / in vivo all 14 subunits are necessary for binding TATA box o TFIIH helicase activity melts the DNA @ start site
Regulation by multiple transcription factors
Pol I, II, and III (general composition and function)
- Eukaryotic Pol’s have 12-16 subunits vs prokaryotic 5 o All Pol’s share 5 subunits
- ONLY Pol II has CTD domain (in its largest subunit) CTD (carboxy terminal domain) composition
- ONLY IN POL II
- Consists of a 7 amino acid repeat sequence
- Phosphorylation of CTD domain is required for Pol II elongation and occurs @ initation
- Phosphorylated CTD also functions as a binding site for mRNA enzymes
Role of CTD phosphorylation in initiation of transcription
- Required for Pol II elongation and occurs @ initiation
- Hyperphosphorylated by TFIIH as Poll II complex moves away from start site
TATA box position and role in positioning pol II for transcription
- TATA box sequence lies 25-35 bp upstream of start site
- Commonly found in HIGHLY TRANSCRIBED GENES
Initiator sequences
- SEQUENCE OVERLAPS START SITE
- Poorly conserved
- Pyrimidine rich
CpG islands
- 60%-70% of genes ARE NOT transcribed as frequently as those that have TATA boxes or initiator sequences
- “House Keeping Genes”
- GC-richs sequences that initiate @ several start sites and in both directions
General transcription factors (TFIID composition, TFII H helicase activity)
- MULTIMERIC PROTEINS
- TFIID- largest TF in Pol II and consists of 1 TBP and 13 TAF’s
- TFIIH has helicase activity and melts DNA at start site using ATP HYDROLYSIS
- TFIIH continues to phosphorylate the Pol II CTD as transcription moves away from promoter
Assembly of pol II preinitiation complexes in vitro
- 1) TBP binds TATA sequence; TBP= monomer, and interacts with the MINOR GROOVE
- 2) TFIIB conacts both DNA and TBP
- 3) Preformed TFIIF-Pol II complex binds and is positioned over start site
- ** Other TF’s must bind before the DNA duplex can be separated to expose the template strand***
- 4) TFIIE (tetramer) binds and creates a docking site for TFIIH
- 5) Binding of TFIIH completes assembly of transcription preinitiation complex in vitro
Pol II pausing and regulation of elongation by NELF and CTD phosphorylation
- NELF (negative elongation factor) complex binds after transcribing about 20-50 nt
- Elongation proceeds after binding of DSIF and specific phosphylation of NELF and CTD domain by TFIIH
Promoter analysis by linker scanning mutation analysis
- Identifies transcriptional control elements in promoter regions
- Promoter regions of any gene can be linked to a reporter gene for analysis (fluorescent tagging)
Pax6 (eukaryotic transcription factor gene and cell type-specific control elements)
- TF’s are regulatory proteins that bind to specific DNA control elements
- Each TF binds to only one specific control element (eg. tissue specific)
Promoters, promoter-proximal elements and enhancers in eukaryotes
- Promoter proximal elements & enhancers = cell-type specific
- Promoter proximal elements = control regions that lie w/in 100-200 bp upstream from start site
- Enhancer is > 200 bp from start site UAS (upstream activating sequence in yeast)
- Function like promoter proximal and enhancers
- Most yeast genes contain only on UAS a few hundred bp from start site
DNase I footprinting * DNase endonuclease cuts DNA fragment at specific points * Binds the minor groove of DNA and cleaves the phosphodiester backbone * Corresponding TF will bind to DNA and protect it from endonuclease cuts * Used to discover the sequence specificity of DNA binding since specific protein binding to DNA will protect is from cleavage
Column chromatography techniques for purifying proteins * Used to separate proteins into different fragments based on size, charge, and hydrophobicity * Once a TF binds the DNA, the DNA-protein complex will move slower during electrophoresis
EMSA (electrophoretic mobility shift assay) * Proteins/TF’s that bind to DNA fragments will move slower on a gel than free proteins * Detects whether DNA sequences are bound by specific proteins
DNA affinity chromatography * Last step in TF purification * DNA control element is covalently linked to the column
Transcription factor modular structure (DNA-binding vs transcription activation/repressor domains) * Transcription-activating function and DNA-binding function are located in different domains called Modular Structure * TF’s contain ONLY 1 DNA-BINDING DOMAIN but can contain >1 TRANSCRIPTION ACTIVATING DOMAINS * DNA-binding domains determine which genes will be transcribed * Transcription activating domains will activate any TF binding domain
Helix-turn-helix motif and recognition helix in bacterial repressor proteins * Helix-turn-helix motif recognized by bacterial repressor proteins * Bacterial repressors and eukaryotic TF’s will bind as DIMERS * Contacts b/w α-helix and major groove determine binding-affinity
Homeodomain
* About 60 amino acids long and similar to helix-turn-helix motif * Most commonly found in TF’s that function during development
Zinc finger domain * Common in TF’s * Cysteines and histidines in the motif bind Zn 2+ via sulfhydryl bonds * C2H2 = most common DNA-binding motif in human genome & multicellular plants * Contains a 23-26 residue consensus sequence w/ 2 conserved cysteine and 2 conserved histidine residues * Binds with major groove as a MONOMER

* C4 is found in some TF’s * Contains a 55 or 56 residue domain that features two groups of 4 critical cysteines * Bind as a DIMERS
C2H2 and C4 zinc finger * C2H2= most common DNA-binding motif * 23-26 residue consensus sequence contains 2 cysteines and 2 histidines * Binds major groove as MONOMER * C4 binds as DIMER * 55-56 residue contains two groups of 4 cysteines leucine zipper proteins * Many proteins contain α helices with hydrophobic amino acid Leu @ every 7th position Coiled-coil motif * α helices have 3.6 residues/ turn, and Leu @ every 7th position so one side of each helix is hydrophobic (amphipathic) * facilitates coiling around each other to form a dimer (like a dimer) * α helices have residues that bind to major groove * bZIP and bHLH can bind as homodimers or heterodimers * Can recognize the same DNA-binding site * Can even bind as heterodimers to different but related DNA-binding sites * ****Different combinations of heterodimers elicits varied responses**
Amphipathic -helix, coiled-coil dimer * Since every 7th position contains a hydrophobic Leu residue, one side of each helix will be hydrophobic = amphipathic * This causes the helices to wrap around each other as DIMERS
Structural properties of activation/repressor domains * Activation domains have High content of acidic amino acids (asp, or are glutamine-rich, or proline-rich * Some activation domains need binding of co-activator proteins to become structured * Others are highly structured but need binding of ligand to change to active conformation
Ligand activation of estrogen receptor and Tamoxifen antagonist * Binding of hormone estrogen to estrogen receptor cause conformational change which promotes binding of co-activator that activates Pol II * Binding of antagonist Tamoxifen will block binding of co-activator, thus preventing transcription by Pol II
Combinatorial regulation of bZIP and bHLH heterodimers * Can recognize the same DNA-binding sites and bind as homodimers or heterodimers * Can recognize different but related DNA-binding sites * Different combinations of heterodimers= varied response

Cooperative binding of transcription factors and enhanceosome * Binding-affinity of TF increases as more TF’s bind to DNA * Complex of cooperative TF’s around an enhancer region = enhancesome
Gene silencing by heterochromatin condensation and role of histone modifications * Condensation readily occurs in TELOMERES AND CENTROMERS where there is NO TRANSCRIPTION * Chromatin condensation promoted by histone deacetylation and/or methylation * Histone deacetylation removes (-) negative charges and promotes histone-histone interactions (favors condensation)
Telomere condensation, Rap1 protein, and deacetylase activity * Removal of histone acetyl groups promotes histone-histone interactions condensation @ telomere * Rap1 proteins bind telomere repeat sequences and recruit Sir protein complex, of which one (Sir2) has deacetylase activity
Promoter-localized chromatin condensation and acetylase/deacetylase activities * A repressor domain can recruit a protein complex that has deacetylase activity localized nucleosome condensation and prevents Pol II from binding to TATA box * An activator domain can recruit a protein complex with acetylase activity localized nucleosome acetylation allowing Poll II to bind to TATA box
Chromatin remodeling factors * Facilitate the “sliding” of nucleosomes on DNA allowing TF’s to bind freely with de-condensed chromatin * Some promoters require chromatin remodeling factors
Mediator complex general composition and interactions * Forms a bridge b/w activator domains and Pol II * Comprised of about 30 subunits, some of which are necessary for transcription of ALL genes * Multiple TF’s bound to promoter-proximal or enhancer regions make contact w/ 1 or more subunits of the mediator complex (require bending of DNA) * TAF subunits of TFIID associate with mediator complex * Absence of mediator complex minimal basal transcription
Lipid soluble hormones and mode of action * Hormones are small enough to defuse through cellular membrane and directly activate TF’s

Common domain structure of nuclear receptors * All nuclear receptors share a common domain structure * Utilize DNA-binding motif C4 zinc finger * Have 1) variable region 2) C4 zinc finger DIMER 3) Ligand binding domain * Heterodimeric nuclear receptors (VDRE, TR, RARE) are EXCLUSIVELY found in nucleus * In absence of ligand they repress transcription by directing histone deacetylation * Homodimeric nuclear receptors (ER, GR) are found in cytoplasm in absence of ligand * Hormone binding leads to translocation nucleus
DNA response elements characteristics *

Epigenetic regulation (DNA methylation, 5-methyl C, histone modifications) * Inherited changed in phenotype that do no result from changes in DNA sequence * Changes in gene expression are initiated by TF’s and are maintained over many cell divisions * Epigenetic control involves histone methylation (5’- methyl cytosine, often in CpG islands) chromatin condensation
Pol I and Pol III promoters * Each has own set of TF’s * Transcription rates are coupled to cell growth and proliferation * TBP = required by ALL 3 Polymerases
Chapter 8
Various RNA definitions (pre-mRNA, hnRNA, snRNA, snoRNA, miRNA, siRNA) * Pre-mRNA= freshly made mRNA w/in the nucleus that has introns and NO poly-A tail * hnRNA= nuclear mRNA that include pre-mRNA and RNA processing intermediates * snRNA= 5 nuclear RNA’s that function during splicing * snoRNA= nuclear RNA’s that direct complementarily bind to regions of pre-mRNA and direct cleavage * miRNA= 22bp long and bind extensively NOT perfectly with mRNA molecules leading to TRANSLATION INHIBITION * siRNA= 22bp long and bind perfectly with mRNA leading to DEGRADATION
Capping reactions and enzymes * 1) after about 25 nucleotide…. 5’ capping of pre-mRNA by several enzymes: * Phosphohydrolase * Guanylyl transferase * Guanine-7-methyl transferase * 2’-O-methyl transferase * All enzymes associate with phosphorylated CTD hnRNPs (heterogeneous nuclear RNPs) and hnRNP protein functions * hnRNPs associated with pre-mRNA in the nucleus * hnRNPs= required for processing and block formation of pre-mRNA secondary structures * some hnRNP’s remain in the nucleus while others shuttle b/w nucleus and cytoplasm (during transport) * possess RNA binding motifs
GU and AG conserved sequences at ends of introns * Splicing relies on GU and AG consensus sequence * “GU”= 5’ * “AG”= 3’ * “A” residue @ branch point about 20-50 base from 3’ splice site, lies upstream of pyrimidine region * All 3 are INVARIANT
A residue branch point in intron and pyrimidine-rich region (approximate distance from intron 3’ end) * “A” residue = branch point, FIRST TRANSESTERIFICATION where 5’ end of intron in linked to 2’ adenosine by phosphodiester bond * A residue forms a “branch” in the lariat structure * 5’-2’ phosphodiester bond
Transesterification reactions in splicing (1st and 2nd) * Transesterification- exchange of one phosphodiester bond for another * 1st transesterification is 5’ phosphate to 2’ OH of adenosine @ branch point * 2nd transesterification is joining of 3’-5’ which releases the “lariat” structure and the remaining snRNPs
Intron lariat structure and 2’-5’ bond * Assembly of the splicesome (U1 @ 5’, U2 @ 3’) catalyzes 1st transesterification reaction that joins 5’ end of intron to the 2’ hydroxyl of A residue, lariat structure is formed * After 2nd transesterification where the 3’ OH and 5’ phosphate ends are joined, the lariat structure is released snRNAs and snRNPs (U1, U2, U4, U5, U6) * over 170 proteins needed for splicing * snRNAs associate with 6-10 proteins to from snRNPs * Short regions of U1 and U2 base pair with consensus sequence at 5’ and 3’ ends, respectively * After U1 and U2 snRNPs have associated with pre-mRNA the trimeric complex of U4, U5, and U6 join the initial complex to form the splicesome
U1 and U2 base-pairing to pre-mRNA, compensatory mutation evidence, and spliceosome * U1 and U2 base pair to consensus sequences at 5’ and 3’ end * Base pair complementation is necessary as shown by mRNA mutations that disrupt base pairs (C-A instead of C-G) and compensatory ones that restore base pairing

Phosphorylated CTD structure and role in posttranscriptional modifications * Phosphorylated CTD associates with capping enzymes, splicing factors, and polyadenylation factors * VERY LONG
Exonic splicing enhancer sequences and SR proteins * Information for defining splice sites is also encoded in sequences of exons called “exonic splicing enhancers” * SR proteins (rich in Ser and Arg residues) bind to exonic splicing enhancers along with snRNPs and splicing factors to form “cross exon recognition complexes” * *****permit precise specification of exon-intron junctions in long pre-mRNAs*****
Self-splicing introns * Some introns can slice themselves out of pre-mRNA * Vast majority of tRNA and rRNA in eukaryotes DO NOT have introns * Secondary structure of self-splicing introns resembles snRNAs in splicesome
AAUAAA polyadenylation signal sequence and CPSF protein * AAUAAA sequences lies 10-35 nt upstream of poly-a site * G/U rich region lies 50 nt downstream of poly-a site * CPSF protein binds AAUAAA and CStF protein binds @ G/U region * RNA is looped due to binding of these proteins and is stabilized by addition of cleavage factors * PAP then binds complex and causes cleavage 10-35 nt downstream of AAUAAA sequence
Coupled cleavage and polyadenylation * PAP cleaves 10-35 nt downstream of AAUAAA * After cleavage PAP synthesize 12 A residues @ SLOW RATE * PABPII binds to the short poly-A sequence and rapidly accelerates the addition of A residues by PAP * Rapid addition of poly-A requires multiple PABPIIs to bind the growing tail * After about 200-250 A’s are add PABPII signals PAP to stop
PAP (poly(A) polymerase) and two phases of poly (A) addition * PAP binds to the complex of cleavage factors and CPSF * PAP causes cleavage 10-35 nt downstream of AAUAAA sequence * After cleavage PAP synthesize about 12 A residues (PHASE 1) * PABPII joins the party and the short poly-A tail and accelerates the addition of A residues by PAP (PHASE 2) * After 200-250 A’s PABPII signals PAP to stop
PABP II (poly(A)-binding protein II) * Binds the short poly-A tail during PHASE 1 and accelerates addition of A residues by PAP * After 200-250 nt PABPII signals PAP to stop

Regulated alternative splicing in Drosophila sex determination (role of Sxl, Tra, and Dsx proteins) * Sxl protein promotes skipping of specific exons * Tra protein promotes joining of specific exons * Sxl accumulates in females early in development and promotes skipping of of exon 3 in its own pre-mRNA * Sxl also promotes skipping of exon 2 in Tra protein * Presence of Sxl and Tra proteins promotes female form of Dsx protein; males lack Sxl and Tra proteins
Dscam gene and riole of alternative splicing in neuron synaptic connections *
K+ channels in hair cells and role of alternative splicing * K+ ion channels open as a response to increased intracellular Ca 2+ concentrations * Each hair cell expresses a mixture of alternatively spliced K+ channel mRNA’s * The gene that encodes for this Ca 2+ activated K+ channel is expressed as multiple alternatively spliced mRNAs * Concentration of Ca 2+ correlates with different sound frequencies
RNA editing * VERY RARE IN HIGHER EUKARYOTES, WIDESPREAD IN MITOCHONDRIA AND CHLOROPLASTS * Ex. apoB gene is part of LDL complex in serum that delivers cholesterol to body tissues * ONLY LIVER CELLS CAN DELIVER but alternative form of the protein exists in the intestine * Intestinal cells have enzymes that change a base in the coding sequence creating a STOP CODON cannot transport cholesterol
Nuclear pore complex, nucleoporins and FG-nucleoporins * NPCs= 30nm in diameter and have a complex shape with 8 filaments that extend on either side of the pore * ALL transport in/out of the nucleus of “stuff” larger than 40-60kda goes through NPCs--------- anything smaller simply diffuses (hormones) * Larger complexes require transporter proteins like importins or exportins to move across nuclear envelope * ***mRNP export is rarely regulate but improperly process mRNA will be degrade by exosomes in the nucleas**** * FG- Nucleoporins line the interior walls of NPCs * FGs= hydrophobic * Have Phe and Gly rich domains * NXF1/NXT1 =heterodimer complex that binds to exon-junction complexes to export mRNP * REF= RNA Export Factor * Hydrophobic regions on surface of NXF1/NXT1 complex interacts w/ Phe – Gly –rich domains of FG nucleoporins

NXF1/NXT1 transporter protein for mRNP * NXF1/NXT1 form heterodimer complex that exports mRNP * Hydrophobic regions on the surface interacts with hydrophobic regions of Phe and Gly domains of FG nucleoporins to facilitate diffusion mRNP remodeling (CBC [nuclear cap-binding complex], and poly(A) binding proteins) * Some mRNPs dissociate before transport and some after transport of mRNA through NPC * mRNP remodeling occurs in the cytoplasm * ** Nuclear CAP binding complex is replaced by eIF4E and PABPII is replaced by PABP1 eIF4E (cytoplasmic cap-binding protein, subunit of eIF4 translation initiation factor) * eIF4E replaces nuclear CAP-binding complex * eIF4E = translation initiation factor
PABPI (cytoplasmic poly(A) binding protein I) * PABP1 replaces PABPII miRNAs (micro RNA) and siRNAs (silencing RNA), properties and mode of action * Short stranded RNAs about 21-26 nt long * Base pair to specific target mRNA’s * miRNA= imperfect matching leading to translation inhibition * siRNA= perfect matching leading to degradation * function w/in cells to block excessive DNA TRANSPOSITION and providing defense against viral infection in plants * Transfection with long dsRNA simulates viral infection in eukaryotes and will induce antiviral defenses miRNA processing (hairpin precursor, Drosha, Dicer * miRNA’s bind at the 3’ UTR of multiple target mRNA’s * a SINGLE miRNA regulate translation of MULTIPLE mRNAs with similar not identical 3’ UTRs * nucleotides 2-7 are most critical for targeting to a specific mRNA
Pre-miRNA processing * pre-miRNA transcripts vary in size and are cleaved in the nucleus to ~70nt by Drosha (dsRNA nuclease) * cleavage of pre-miRNA’s to 70nt forms a “hairpin” structure * some miRNA’s are processed from introns and 3’ UTRs of pre- mRNAs * hairpin structure is then exported from nucleus * 70nt pre-miRNA is then bound to Dicer + TRBP (binding protein) * Dicer cleaves the 70 nt hairpin 21-23 nt long w/ unpaired nt at each end * RISC complex targets the complementary strand to mRNA and the other is degraded * Argonaute protein w/in RISC in CRUCIAL for its function of inhibiting translation * RISC binds mRNPs @ 3’ end
RISC (RNA-induced silencing complex), Argonaute subunit function * RISC will bind 21-23 nt long pre-miRNAs after cleavage by DICER * RISC targets the strand complementary to the target mRNA and Argonaute protein binds the target strand thus inhibiting translation cytoplasmic polyadenylation in fertilized eggs (CPE, CPEB, maskin, role of phosphorylation, CPSF) * Stored mRNAs contain very short poly-A tails (20-40 residues) that DO NOT bind PABP1 no translation due to necessary interaction b/w eIF4E, Eif4G, and PABP1 * Stored mRNAs have a cytoplasmic control element (CPE) upstream of the AAUAAA poly-A signal sequence in the 3’ UTR * CPEB binds to CPE and associates with Maskin protein which in turn binds to eIF4E thus preventing binding w/ eIF4G * Fertilization promotes CPEB phosphorylation and dissociation from Maskin * CPSF (Cleavage and Polyadenylation Specificity Factor) binds to poly-A site and dephosphorylated CPEB * PAP is recruit for addition of A residues then followed by PABP1 binding to poly-A tail role of eIF4G and PABP in translation * Multiple PABP1 proteins bound to poly-A tail of mRNA interact with eIF4G initiation factor * This stabilizes the interaction of the 5’ cap with eIF4E CAP-binding protein * PABP
Pathways of mRNA degradation (deadenylating enzyme, decapping enzyme, P-bodies)
Exosome (complex of 3’ to 5’ and 5’ to 3’ exonucleases) and nucleus)
AU-rich sequences in short-lived mRNAs
Iron-dependent regulation of ferritin and transferring receptor mRNA translation (IRE, IRE-BP)
Role of mTOR and eIF2 kinases in translation
Nonsense-mediated decay of mature mRNAs (exon-junction complexes, Upf1, P-bodies) mRNA localization rRNA processing (transcription unit, role of snoRNPs, methylation, pseudouridylation)
Ribosome assembly (pre-90s, pre-60S) tRNA processing ((RNase P, CCA sequence, small intron