Molecular Genetic Final

rRNA splicing

mature rRNA cleaved from longer pre-rRNA with endo and exoribonucleases

endonuclease III cleaves double stranded RNA (related to eukaryotic dicer), ribozyme P cleaves near 5S, exonucleases trim ends after to produce mature rRNAs

tRNA processing

RNase E/F endonuclease cleaves at 3’ hairpin → RNase D exoribonuclease cuts blunt end → RNase P cleaves at 5’ hairpin → RNase Z endonuclease cleaves 3’ extension → followed by synthesis of 5’CCA3’ end of tRNA nucleotidyltransferase

tRNA modifications increase flexibility of tRNA to interact with different codons, allows aminoacyl tRNA synthetases to recognize their cognate tRNA family (6 tRNAs) to connect to correct aa to carboxyl group 3’ of tRNA

prokaryotic mRNA processing

intrinsic termination hairpin prevents degradation of mRNA beginning at 3’ end by RNase II and PNPase, RNase E and RNase III remove hairpin to allow RNA to be degraded by degradosomes

eukaryotic transcription

after preinitiation complex from promoter region formed RNAPII moves down making mRNA transcript, mRNA capping required - adding GTP to 5’ end

capping

5’ triphosphate of GTP with 5’ triphosphate of mRNA, PPi released, cap added, additional methyl caps from S-adenosyl-L-methionine added

functions as promoter escape by RNAPII, transport of mRNA to cytoplasm, initiation of translation, stability of mRNA

polyadenylation in transcription termination

polyA signal on pre-mRNA AAUAAA where CPSF (cleavage polyadenylation specific factor) binds, which is bound to PolyA polymerase with polyadenylate binding protein and CstF (cleavage stimulation factor) creating PolyA tail on 3’ end

CPSF part of RNAII elongation complex, attaches to polyadenylation signal sequence so termination favored over elongation

functions include transcription termination, mRNA processing, transport of mRNA to cytoplasm, mRNA stability, mRNA translation

not all mRNA transcripts polyadenylated (like histones - 3’ end formed by endonucleolytic cleavage)

GU-AG introns

key role in splicing reactions, consensus sequences include 5’ splice site (GU), polypyrimidine tract, and 3’ splice site (AG)

transesterification reaction

2 occur in splicing pathway so no change in number of phosphodiester bonds

3’ hydroxyl group of branch site attacks donor site forming lariat → exons join at acceptor site, lariat unbranched and degraded

accurate splicing

spliceosome needed to ensure proper splicing, has snRNPs where each hairpin forms binding site for different protein

exon skipping joins exons not adjacent, cryptic splice site selections joins part of exon to another exon

intron removal

U1-snRNP binds 5’ end of intron through RNA pairing, U2-snRNP binds at SF1 and attraction between these proteins pulls them together, U4/6 and U5-snRNP bind and spliceosome

SR (serine arginine) protein

bind on sequence on exonic splicing enhancer on exon to define location of exon for spliceosome

Sex determination in Drosophila

determined by DSX protein’s 2 alternatively spliced forms, SXL redirects splicing of TRA which redirects splicing of DSX

sxl - males have 2,3,4 spliced together with no SXL made (truncated protein), females have 2 and 4 spliced so SXL made

tra - males have U2AF bound so 1 and 2 and no TRA, females have SXL blocking U2AF so 1 and part of 2 spliced and TRA made

dsx - males have 3 and 5 spliced so male specific DSX made, females have TRA on 4 allowing SR proteins to bind so 3 and $ splice (not 5 because intron have no GU sequence) and female specific DSX made

trans-splicing

roundworm RNA cant be initiated by ribosomes in translation, spliced leader RNA (SL-RNA) spliced to target RNAs make them translatable to combine exons from different transcripts by providing attachment point for ribosomes, clustered genes transcribed into one polycistronic mRNA

hydroxyl attack from branch site to donor site, exon joins other exon at acceptor site but no lariat formed

tetrahymenal rRNA splicing

autocatalytic (group I type), ribozymes, pre-rRNA has free G nucleotide that does hydroxyl attack on 3’ end of first exon and then starts intron that circularizes and degrades

deadenylation dependent capping

removal of PolyA tail → decapping → Xrn1 exonuclease digestion of mRNA

life of mRNA limited because each round of translation initiation leads to shortening of tail

nonsense mediated decay (NMD) or mRNA surveillance

can locate incorrect termination codons causing mRNA degradation

exon junction on exon-intron boundary after intron removed → UPF protein binds in cytoplasm → if nonsense codon 5’ of exon junction it is recognized as incorrect so ribosome does not continue and signals degradation

RNA silencing through co-suppression (RNA interference)

adding endogenous promoter with reverse orientation of transgene promoter on other side inserted CHS gene, creates antisense mRNA that binds with mRNA to crease double stranded RNA that trigger RNA interference (white) instead of producing chalcone synthase (purple)

RNA silencing as protective mechanism against ds RNA viruses

dicer nuclease cuts dsRNA virus into siRNA pieces → single stranded siRNA separate and attach to mRNA which is degraded by RISC

RNA silencing through miRNA regulating gene expression

foldback RNAs cut by Drosha (RNA endonuclease) into miRNA precursors in nucleus → cut by dicer in cytoplasm into miRNAs to silence endogenous mRNAs, multiple miRNAs attach to 3’ untranslated region from target RNA → cleavage removes poly A tail leading to no translation initiation or degradation by deadenylation-dependent decapping

genetic code

tRNA is adapter that converts nucleotides to amino acids, groups of 3 because 4³ = 64 possible codons (more than the 20 amino acids)

isolated ribosomes translate mRNA - synthetic RNA polyU tested against all 20 amino acids separately and Phe was incorporated (polyC=Pro, polyA=Lys, GGG=Gly

PNPase 3’→5’ exo-RNase from E.coli

tRNA

informational adapter with bottom connected to codon on mRNA and top connected to corresponding amino acid for peptide chain

20 amino acids interpreted by up to 61 tRNAs (redundancy), tRNAs have common structure to translate codons by specifically charging them with correct amino acid

tRNA structure

3’ end of acceptor arm is where aminoacylation occurs (addition of amino acid), opposite from anticodon where binding to codon of mRNA occurs

TpsiC arm has pseudouridine, V loop variable, D arm has dihydrouridine

base pairing between 3 ribonucleotides in loops cause structure to fold onto itself (upside down L)

amino acid tRNA binding

2’ or 3’ hydroxyl of tRNA and carboxyl of amino acid bind through condensation reaction and release water

uses aminoacyl-tRNA synthetases (aaRSs) which are ligases, glutamate enzyme called glutamate-tRNA^Glu ligase, can recognize up to 6 different tRNAs

aaRS exceptions

bacillus subtilis - no gene for glutamine-tRNA^Gln ligase so it contains a Glu-tRNA^Gln amidotransferase that glutamine directly on tRNA

prokaryotic mitochondria and chloroplasts - translation initiated at AUG MET codon decoded by fMET not MET

wobble

position 34 on 5’ end angle increases base pairing possibilities with third base in codon

wobble rules - A tRNA 5’ anticodon with U mRNA 3’ codon, C→G, G→C or U, U→A or G, I→ A or C or U

G-U base pairing

some tRNA can base pair with 2 different codons, only 2 tRNAs required to encode 4 Ala codons with last position on tRNA being G or U but still coding for alanine

causes problem where tRNA binds to isoleucine codon instead of Met but solved by deaminating at 34 in isoleucine - UAA modified by inosilation to UAI so it can’t decode Met

ribosomes

catalyzes formation of peptide bond between amino acids, places mRNA aminoacyl-tRNA, and translation factors in correct positions

have large and small subunits (80S in eukaryotes and 70S in bacteria), translation initiation by 16S rRNA on small subunit and ribozyme peptidyl transfer by 23S rRNA on large subunit - measured by how fast they sediment in gradient of sucrose centrifuged

A site in middle of subunits where tRNA binding site between both subunits

ribosomes function in vitro so can be reassembled from component parts, protein binding sites on rRNA can be mapped by hydroxyl probing Fe(II), big and can be photographed, X-ray crystallography

translation initiation

ribosomes translate mRNA 5’→3’, produce proteins from N to C terminus

A-site (aminoacyl), P-site (peptidyl). where amino acid added, and E-site where deacylated tRNA leaves ribosome

ribosome binding site

positions ribosome with initiation site in P site, found 3-10 nucleotides upstream initiation codon, where 16s rRNA base pairs

if RBS differs from consensus it will be a weaker site for translation initiation (variation determines translation efficiency)

RBS positions small subunit placing AUG in P site and second codon in A site using IF-3 (prevents premature reassociation of the large and small subunits of the ribosome) → initiator tRNA directed to correct position using IF-2 and GTP hydrolysis (ternary complex) → large subunit joins with help of IF-1

polarity

usully each ORF has own RBS but sometimes translation of ORF2 depends on translation of ORF1

nonsense mutation in first cistron causing lack of translation of 2nd cistron because no RBS so initiation cannot occur, effect of mutation at distant site and can be predicted by overlapping initiation codons, seen in prokaryotes

attaching preinitiation complex to mRNA

elF-2 binds to initiator tRNA within ternary complex component of preinitiation complex, elF-3 makes direct contact with elF-4G (coordinator) and forms link with cap binding complex, elF-4A helicase mediates scanning, elf-4E binds CAP

complex knows when it reaches initiation codon by recognizing Kozak sequence - elF-4B scanning

internal ribosome entry sequence (IRES)

mRNAs that are not capped have this sequence that directly binds to 40S subunit, positioning initiator codon in P-site

found in some host mRNA allowing them to be translated when CAP binding complex blocked through heat stress, anoxia, irraditation, includes IgG heavy chain and Antennapedia

translation elongation

entry of aminoacyl-tRNA into acceptor site with EF-1A (Ef-Tu) and GTP hydrolysis → EF-1A-GDP ejected and peptide bond made at same time acyl bond to initiator tRNA broken with EF-2 and GTP hydrolysis (translocation)

peptide bond formation occurs when amino group of aminoacyl tRNA attacks acyl bond of initiator tRNA

EF-1B never contacts ribosomes but helps regenerate EF-1A-GTP in elongation

CCdA-phosphate-puromycin binding site

23S rRNA catalyzes peptide bond formation - translation inhibitor that binds to A site helped find location of active site, active site of ribosome too far from ribosomal proteins so they can’t catayze reaction

puromycin resembles aminoacyl-tRNA but has no attachment point for codon

eEF-2

eukaryotic counterpart of EF-2, sensitive to diphtheria toxin, a ADP-ribosyl transferase that uses NAD to modify a specific histidine in eEF-2 to inactivate it

diphtheria toxin catalyzes many reactions and eEF-2 inactivated stops translation elongation

translation termination

termination codon enters A site, RF1 and 2 (release factors) recognize termination codons, RF3 stimulates dissociation of RF1 and 2 after termination, GTP hydrolysis occurs to GDP → ribosome recycling factor dissociates ribosome subunits after translation terminated also using GTP hydrolysis

autoregulation in prokaryotes

L11 and L1 large subunit proteins encoded on same mRNA and independently translated, when abundant L1 binds to hairpin at translation initiator site of L11 sequence blocking translation of both

autoregulation in eukaryotes

tubulin monomers assemble into microtubules, excess tubulin binds to site on 5’ UTR of tubulin mRNA to block translation

regulation by iron response elements

ferritin binds and stores iron, iron response protein blocks translation of ferritin → when free Fe binds to 5’ IRP it is released and translation of ferritin mRNA occurs

transferrin pumps iron into cell → when free iron high, Fe binds to 3’ IRP and releases it, causing degradation of transferrin

frameshifting

requires ribosome to pause at frameshifting site allowing tRNA and codon to dissociate, caused by downstream hairpin, RBS-like sequence upsteam, and weak base pairing at AAA

programmed in dnaX mRNA with DNAPIII encoding tau and gamma subunits, both proteins start same but one produced by frameshifting bypassing original stop codon

slippage is overlapped codon reading, bypassing is dissociation of P site tRNA before stop codon → scanning causing slippage without P site tRNA base pairing → pairing p site tRNA with new codon downstream

transient regulation - signal activator/repressor

nonpermanent changes, seen in single celled organisms with nutrient availability and temperature

lactoferrin is protein that binds to reduce Fe in milk, can starve organism of Fe nutrient, activator of transcription of immune genes

transient regulation - signal directly influences protein factor

low Cu in yeast necessary to bind to active Mac1p to make copper uptake proteins, but if Cu too high it binds to Ace1p and copper detoxification proteins made

steroid hormones bind to their receptors and change conformation after binding and enter nucleus to activate transcription

transient regulation - signal indirectly influences protein factor

Lac operon - lacO ahead of RNAP binding site where lacI (allolactose) repressor protein binds so no transcription occurs (RNAP blocked)

cap operator behind RNAP binding site, CAP protein binds at capO for production of full lac operon transcription, CAP activation requires absence of glucose

IIA^Glc is membrane receptor that senses glucose, when glucose transport high it becomes dephosphorylated and inhibits adenylate cyclase and no CAP made, when glucose low it is phosphorylated and ATP used to make cAMP which activates CAP which transcribes lacZ

tyrosine kinase receptor

activates intracellular proteins by tyrosine phosphorylation, STAT (signal transducer and activator of transcription) is directly activated through tyrosine phosphorylation when signal binds to receptor, STAT moves to nucleus and activates target genes

tyrosine kinase associated receptor

activates intracellular proteins indirectly, after signal binds JAK is stimulated to phosphorylate STAT which moves to the nucleus and activates target genes

MAP

mitogen activating protein kinase, MAPkinase is Ser/Thr kinase that activates intracellular proteins by ser/thr phosphorylation

signal binds to mitogen receptor and phosphorylates it → Raf binds to protein and signals transduction → Mek → MK low activates Elk-1 and c-Myc stimulating cell division and if MK high Rsk made which activates SRF

RAS system

signal binds to tyrosine kinase receptor and phosphorylates → GTPase activating protein (GAP) creates inactive Ras with GDP, GNRP creates active Ras with GTP producing Raf and MAP kinase pathway

mating type switching in yeast

permanent change, 12 aa pheromone released by cell to receptor of another cell causing MAP kinase signal cascade to nucleus to differentiate into gametes, and pheromone sent back too so gametes made and fuse into zygotes

relies on genome rearrangement - MAT locus specifies mating type and flanked by silent mating type genes, no mating pheromone creates double stranded break changing allele on gene

immunoglobulins

antibodies produced by B cells, light chain coded by chromosome 2 and has 2 loci with similar arrangements, heavy chain locus on chromosome 14, genes encoded by gene segments

rearrangements mediated by RAG1 and RAG2 recombinases to create different immunoglobulins through alternative splicing

cI - lysogeny

cI gene found between PL and PR promoters and transcribed from PRM, cI repressor binds to PL and PR so cI expression stimulated but blocks early gene expression

CRO gene found after PR, CRO repressor binds to PRM so no cI made but CRO is made

cII with promoter PRE and cIII establish lysogeny

sporulation

prespore forms from mother cell and becomes enclosed spore that is released

sigma A and H regular sigma factors, E and F recognize sporulation gene promoters and not made until signaled by nutrient conditions, SpoOA master switch transcriptional activator through phosphorylation that becomes activated in vegetative cells when signals

sigma F gene activation in prespore through release of SpoIIAB, causes inactivation in mother cell, sigma E determines mother cell with R signal and GA receptor

position effect - vulva

development of vulva cells - 22 cells positioned precisely relative to eggs in 7 8 7 pattern, anchor cells releases LIN3 signals that determine how many cells produced so primary has high conc (8) and secondaries on both sides have low conc (7) through Let-23 receptor that activates MAP-kinase

development of flies - polarity

discrete segments in between head and tail

syncytial embryo lined with nuclei that become own cells in cellular blastoderm, nurse cells are maternal cells that produce bicoid cells that are transported into egg cells before fertilization, bicoid gradient created after fertilization with more bicoid conc towards the nurse cells

mutant embryo has two tails and no head