Genome maintenance Flashcards

Question

What are the roles of TBP?

Answer 1

TATA binding protein - provides sequence specific protein-DNA interaction, bends DNA - recruits other factors (TAFs) in TFIID to promoter region to help w/ transcription stimulation - helps recruits TFIIA + TFIIB to determine positioning of RNA Pol

Answer 2

TFIIF: - stabilises RNA pol interaction w/ TBP-TFIIB - blocks non-specific binding of pol II to DNA - attracts TFIIE & TFIIH TFIIE: - attracts + regulates TFIIH

Answer 3

- has ATP-dependent helicase to unwind DNA - polymerase II C-terminal kinase activates pol + releases it from promoter * has serines at pos 2 & 5 in eukaryotes which can be phosphorylated to activate polymerase.

Answer 4

- phosphorylation of of Pol II CTD by TFIIH CTD consensus is 7 amino acids (ser5 phosphorylated) - TFII (D, A, B + E & H-kinase) dispensable for elongation. - other proteins associate w/ TFIIH to form repairosome ^ defects in this function responsible for Xeroderma Pigmentosum

Answer 5

After promoter clearance, pol II pauses so capping complex is recruited to prevent RNA degradation. Elongation may abort or progress transcription (CTD additionally phosphorylated at ser2 by CDK9)

Answer 6

RNA pol II arrives at poly-A signal (PAS), 3'-TTATTT-5' -> adds 5'-AAUAAA-3' to mRNA Cleavage site 15-30nt downstream of the PAS. RNA pol II adds A bases to 3' end protecting it More efficient if also a U-rich upstream signal element (USE) + GU-rich downstream signal element (GSE)

Answer 7

Consensus sequence analysis if you know the DNA sequence. - purify protein + add to known DNA sequence that has been 5' end radiolabelled - run protein-DNA mix on native gel - DNA bound to protein will be in higher mol weight complex + run slower in gel

Answer 8

DNA sequences in promoters and enhancers of a gene. Have a modular structure.

Answer 9

4 superfamilies

Answer 10

* Activation domains * Repression domains * Domains to interact with other TFs

Answer 11

* Monomers * Dimers

Answer 12

Helix-Turn-Helix (HTH) e.g. homeodomain (HD) Zinc finger (ZF)

Answer 13

Basic-Leucine-Zipper (bZIP) Basic-helix-loop-helix (bHLH)

Answer 14

A structural motif where Helix 3 binds the major groove and Helices 1 & 2 stabilize the structure. The N-terminal arm contacts the minor groove. ## Footnote Example includes the homeodomain family.

Answer 15

Three Class 1 zinc fingers bind DNA, with amino acids within the fingers generating binding specificity. ## Footnote This specificity is critical for transcription factor function.

Answer 16

Found within nuclear hormone receptor family. They are a large superfamily of ligand-dependent transcription factors and receptors. ## Footnote Examples include nuclear estrogen receptors ERα and ERβ.

Answer 17

Yes, they can bind as homodimers (αα or ββ) or as heterodimers (αβ). ## Footnote This flexibility allows for diverse regulatory mechanisms.

Answer 18

Similar structure to bZip. H-L-H domains interact, so basic regions can then bind DNA + target DNA sequence CAnnTG. Many are heterodimers that use a common type-1 partner. There is DNA specificity due to type-2 variable partner.

Answer 19

Muscle - MyoD, activates genes to make muscle cells Neurons - Atoh1, activates genes to make neuronal cells

Answer 20

CLOCK-BMAL1 (helix loop helix heterodimer) binds E-box enhancer sequences in circadian genes (CACGTG) ## Footnote This binding stimulates the transcription of circadian genes.

Answer 21

Gln transactivation domain. PolyQ interacts w/ general factors. ## Footnote Pol II stimulated to transcribe gene

Answer 22

1 linker histone (H1) and 8 core histones (2 of each: H2A, H2B, H3, H4) Central H3-H4 tetramer which 2xH2A-H2B dimers then bind. ## Footnote Nucleosomes consist of DNA wrapped around these histones.

Answer 23

2 turns of DNA. Packaged into 3 nucleosome wide structure ~30nm ## Footnote This structure is crucial for DNA packaging in the nucleus.

Answer 24

Central helix is histone fold domain - where histones bind each other. e.g. H2A-H2B dimer binds H3-H4 N terminal tails exposed + have highly basic residues -> modified for chromatin remodelling. ## Footnote These helices allow histones to bind to one another.

Answer 25

* Methylation of Lysine (K) and Arginine (R) * Acetylation of Lysine (K) * Phosphorylation of Serine (S), Threonine (T), and Tyrosine (Y) * Citrullination of Arginine (R) * Ubiquitination of Lysine (K) * Sumoylation of Lysine (K) ## Footnote Post-translational modifications significantly influence chromatin structure and gene expression.

Answer 26

* Acetylation of Ks in H2A, H2B, H3 & H4 * Methylation of Ks & Rs in H3 + H4 ## Footnote Acetylation and methylation play crucial roles in regulating transcriptional activity.

Answer 27

Occurs in situ (whilst DNA bound) + correlates w/ functional status of relevant genomic locus. Changes define epigenetic 'histone code' -> defines binding potential of chromatin associated factors. e.g. H3-K18 can be trimethylated + acetylated ## Footnote The histone code is a set of modifications that influences gene expression and chromatin structure.

Answer 28

HATS add acetyl groups (CO-CH3) to histones ## Footnote Examples include yeast Gcn5, CBP, PCAF, and TAF250.

Answer 29

HDACs remove acetyl groups from histones ## Footnote Examples include yeast Rpd3 and human HDAC1.

Answer 30

Acetylation is associated with transcriptional activation ## Footnote It reduces charge, leading to less cross-binding of histone proteins and a relaxed chromatin state.

Answer 31

CLOCK has HAT-activity, acetylating H3 and H4, which opens up the core tetramer for DNA accessibility ## Footnote It also relaxes chromatin and recruits TFI and Pol-II. Keeps basal promoter region open for TFII + Pol-II

Answer 32

p300/CBP can interact with various transcriptional regulators like pCAF (HAT)& TBP. Acteylation by p300/CBP & TAFII250 facilitates the assembly of the pre-initiation complex -> driving transcription. ## Footnote It helps drive transcription by recognizing the promoter TATAA.

Answer 33

p300/CBP & TAFII250

Answer 34

Enzymes that add methyl groups to lysine residues

Answer 35

Remove methyl groups from lysine residues

Answer 36

Transcriptional repression | 1, 2 or 3 methyl groups can be added to NH3+ residues on Lys. Replaces P

Answer 37

SUV39H1 and SUV39H2

Answer 38

Transfer of methyl groups from S-adenosylmethionine (AdoMet) to arginine residues

Answer 39

Only one, JMJD6

Answer 40

Transcriptional activation ## Footnote e.g. PRMT1 methylates H4R3

Answer 41

Methylates H3R17 + non-hisotne protein p300/CBP triggering its HAT activity for transcription

Answer 42

Chromatin function by driving association with specific bound regulatory proteins ## Footnote For example, H3K4me3 & H3K9me3 bind to PHD finger domain proteins like ING2 and BPTF.

Answer 43

Chromodomains - HP1 ## Footnote These modifications are associated with heterochromatin formation.

Answer 44

Bromodomains - PCAF ## Footnote This binding is significant for chromatin acetylation and gene activation.

Answer 45

Controls transition between chromatin states. They speed up histone sliding to reveal DNA binding sequence -> TFs can recruit HATs to fix chromatin in active state. ## Footnote They include ATP-dependent chromatin remodeling and histone acetylase/deacetylase containing complexes.

Answer 46

* ATP dependent chromatin remodeling histone sliding (e.g., SWI/SNF, NURF) * Histone acetylase/deacetylase containing complexes (e.g., SAGA, PCAF) ## Footnote These complexes facilitate the dynamic changes in chromatin structure.

Answer 47

Active gene expression ## Footnote Euchromatin is less condensed and accessible for transcription.

Answer 48

Generally inactive ## Footnote Heterochromatin is more condensed and less accessible to transcription machinery.

Answer 49

Removes H2A-H2B dimers ahead of polymerase -> histones disassemble ## Footnote This allows Pol II to advance and transcribe the DNA.

Answer 50

FACT helps reassemble histones to form new nucleosomes further upstream, towards 5' end ## Footnote This process is crucial for restoring chromatin structure after transcription.

Answer 51

Enhancers are regulatory elements that work at a distance to activate transcription by facilitating the recruitment of the transcription pre-initiation complex ## Footnote They enable DNA looping and drive efficiency in forming the pre-initiation complex.

Answer 52

* Proline-rich * Glutamine-rich * Acidic (glutamate + aspartate rich) ## Footnote These domains interact with other proteins to activate transcription.

Answer 53

LCRs act like enhancers but function over longer distances and have multiple binding sites for TF complexes to activate multiple linked genes ## Footnote 12 LCRs have been identified in humans so far.

Answer 54

Insulators provide boundaries for domains within euchromatin and prevent unwanted interactions between chromatin regions ## Footnote They stop enhancers from regulating incorrect genes.

Answer 55

Barriers are more important at the boundary of heterochromatin and euchromatin, preventing heterochromatin from spreading into gene-containing regions ## Footnote Insulators primarily define interactions within euchromatin.

Answer 56

Nucleosomes are fundamental structural units of chromatin ## Footnote They consist of DNA wrapped around histone proteins.

Answer 57

Chromatin folds and loops to form discrete chromosome territories, with euchromatin and heterochromatin occupying different zones as CTs are polarised. ## Footnote This organization is crucial for gene regulation and expression.

Answer 58

False ## Footnote Core promoters are distinct regions that initiate transcription, while enhancers are regulatory elements that enhance transcription from a distance.

Answer 59

N-terminal tails which are exposed + have highly charged basic residues. - essential for chromatin remodelling

Answer 60

Genome regions can be gene rich or poor. Different chromatin domains fold into distinct 1Mbp DNA foci in situ. Organise eukaryotic chromosomes - compact chronatin fibers. ## Footnote active + inactuve hgiher order domains separated

Answer 61

Used Br-UTP (green) Found mRNA synthesis sutes clustered in 'factories' that have many genes w/ 5-10 active RNA pol-II complexes

Answer 62

3 NF-kb target genes in same region in HUVEC cells following TNF treatment. Co-localised + recruitment is ordered ## Footnote tertiary trasncription complex provides synergistic co-regulation

Answer 63

- Secreted ligands (morphogens) released from a source (organiser) signal to nucleus to drive tissue specific signalling pathways - Extracellular morphogens lead to intracellular TF gradients that drive alternative transcriptomes in cells - TFs function at different thresholds to regulate genes via a digital (OFF-ON) and/or analogue (GRADED) level to drive cells to different fates.

Answer 64

Involves transmembrane or nuclear receptors. Ligand binds receptor + either: - causes change in conformation -> signal transduction via phosphorylated 2nd messenger (becomes TF) - ligand is steroid hormone so is internalised at nucleus (becomes TF)

Answer 65

TFs either increase or decrease transcription - driven by basal machinery. One TF can drive multiple genes to be upregulated -> cascade

Answer 66

Master TF preferentially recognises 5'-ATTTAAAT-3' in enhancer regions. MTF can bind 5 similar sequences present in 20 genes. Primary genes (A1, B6, C11)-> TF proteins Secondary genes indirectly transcribed -> TF proteins ## Footnote e.g. secondary protein D21 is an activator of tertairy genes (G41-45)

Answer 67

Extracellular conecnetration of morphogen. Intracellular MTF conc dircetly correlates w/ conc of morphegen received. So cells nearest morphogen source generate most MTF - due to graded level of signal transduction factor (morphogen). MTF conc leads to differential transcriptomes & alternative cell fates. ## Footnote A single TF can drive alternative transcriptomes at different concs.

Answer 68

Will change the cell if it is present above a threshold. e.g. any cell w/ > 25nM MTF will convert from fate X to Y Switch occurs as at the threshold, gene Y can activate (auto-regulate) its own expression. ## Footnote Once upregulated, cell has Y-competence so Y fate will persist even if MTF removed.

Answer 69

Morphogen gradient cna also provide an anlogue signal to sub-pattern Y cells. e.g. 25nM = Ya, 30nM = Yb, 35nM = Yc at low concs, only highest affinity targets bound by MTF at high cocns, MTF activates lowest affinity sites, excess mols can bind further sites, ## Footnote higher the ceoncnetration, more genes bound so much ahrder to spot lower affinity genes

Answer 70

lower affinity genes often codes for repressor for a higher affinity gene (e.g. B7 for A genes) Single TF can generate cells w/ very different transcriptomes due to alternate casacde triggered ## Footnote BUT different fate of low affinity gene could be due to expression of additional genes

Answer 71

Segmentation genes provide AP identity within each individual segment (polarity) Patterning genes provide unique segment identity along AP axis ## Footnote Patterning driven by initial maternal bicoid mRNA concentration gradient.

Answer 72

- high conc bicoid protein activates zygotic hunchback (anterior) - hunchback gradient est domains exprssing distinct GAP genes - hunchback combines w/ GAP genes to co-activate pair-rule genes | GAP genes - kniros, tailless, giant, knippel pair rule genes - even-skip ## Footnote Pair rule gens activate segementation genes (wingless/hedgehog) hunchback, GAP + Pair rule TFs combine to co-activate patterning genes (Hox)

Answer 73

Ubx mutant cuases T3 to turn into T2 - complete w/ wings instead of halteres. -> master AP-patterning genes are homeobox (Hox) family Same in mice but they have multiple Hox genes | Lewis in 1970s ## Footnote All patterning genes driven by concentration of retinoic acid

Answer 74

Initial signalling generates broad fate restriction. e.g. endoderm, mesoderm, ectoderm + neuroectoderm. Gradients then re-used to generate sub-fates -> depends on precise time, conc + order in which it receives key morphogens

Answer 75

Competition between 4 morphogen gradients - TGF, BMP, FGF & WNT. -> determine swhich MTFs will drive transcriptome ## Footnote these 4 pathways + their interactions generate all possible trasncriptome combinations -> all diverse cell types

Answer 76

In most prokaryotes, factors bind single specific OriC site to form replication bubble. Easily unwound DNA is A-T rich (weaker) - broken due to mechanical twisting of DNA by DnaA - DnaB (helicase) is loaded + binds both strands by DnaC (helicase loader) - primase can use DnaB as reference point for specific pairing ## Footnote DNA synthesis semi-conservative (Meselson & Stahl) - parent strand used as template.

Answer 77

dNTPs have triphopshate + hydroxyl end - nucleotide monophosphate added to chain w/ release of diphosphate. Synthesis only in 5'->3' direction

Answer 78

2 growing forks at once in 'replication bubble': Leading strand (5'->3') is continuous Laggin strand (3' -> 5') is discontinuous, added in steps

Answer 79

- Primase synthesises short RNA primer - DNA Pol III extends primer into Okazaki fragment - DNA Pol I can displace RNA primer in the way (5'->3' exonuclease activity) + add bases - DNA ligase joins adjacent nucleotides w/ phosphodiester bond ## Footnote some DNA polymerases have 3'->5' exonuclease activity (proofreading)

Answer 80

Dimeric - has monomers associated w/ leading + lagging strand. - a-subunit is polymerase centre - clamp loader grabs beta subunit which recognises DNA/RNA hybrid, csliding clamp locked over primer - a-subunit binds clamp + synthesisies strand ## Footnote exonuclease activity in palm shape area of a-subunit

Answer 81

Hexameric - catalyses unwinding of DNA duplex to expose single strand DNA - Different helicases w/ different polarities on 2 strand in duplex - ATP hydrolysis provides energy for unwinding

Answer 82

Single stranded protein - tetrameric Binds ssDNA to prevent duplex reannealing - >1000 fold afffinity for ssDNA over dsDNA Lowers DNA melting temp promoting denaturation Exhibits co-operative binding.

Answer 83

Initiates okazaki synthesis on ssDNA template - displaced by DNA Pol III after 10-12 ribonucleotides added - Pol III synthesises from 3'OH groups on RNA primer Forms complex w/ helicase in lagging strand synthesis

Answer 84

Lagging strand synthesis - 5'->3', catalyses chain elongation by formation of phosphodiester bonds - only extends chains from 3'OH termini, cannot initiate synthesisi of new chains - primer removal in lagging strand synthesis has both 3' (proofreading) and 5' (primer excision) exonuclease activity

Answer 85

180 degrees from OriC are Ter sites (replication fork traps) - 2 sets for both directions of DNA synthesis - - binding sites for Tus (DNA binding protein) as a monomer Can also directly inhibit replication fork progression by contacting DnaB (inhibiting unwinding)

Answer 86

- RNA primer extended by DNA pol alpha - Pol delta + helicase push aside primer to extend strand - Fen1 (endonuclease) cuts at branchpoint /flap at 5' end DNA - DNA ligase links adjacent fragments

Answer 87

DNA between adjacent termini, can replicate independently with its own origin site. Almost all replication units are bidirectional. Eukaryotes have fragmented genome (multi-chromosomal) so multiple units of DNA replication needed

Answer 88

Origin defined by complex 3D combination of chromatin features that generate ORCs (origin recognition complexes) origin function is redundant - more origins than necessary -> stochastic component to origin selection - no need for specific sequence ORCs give platform for assembly of pre-initiation complex - recuited in cell-cycle dependent manner (controlled by cyclin/CDK) -> proteins only bind ORCs in window of opportunity to form multi-complex

Answer 89

S. cerevisiae - 1st replication origins to be characterised. Discovered autonomous replicating sequences (ARS) - contains 3 essential conensus domains (A had high T-A) - bound by ORC complex

Answer 90

CLB6 - acts in early S-phase, rapidly degrades CLB5 - operates late in S phase *in yeast ## Footnote each origin has an innate timing preference

Answer 91

Competency is lieklihood of ARS being used to replicate DNA Some ARS elements very strong, others very weak

Answer 92

AT rich sequences w/ no clear consensus - defined by local chromatin context.

Answer 93

- Cdc6 - highly unstable, synthesised during G1, binds ORC + recruits Cdt1 - Cdt1 also only expressed early in G1 - it loads McM to establish pre-RC complexes BUT Geminin expressed late G1 + S phase - blocks Mcm loading ## Footnote If factors are loaded, then that region is licensed to duplicate DNA during that cell cycle

Answer 94

Takes 9-10 hours - Euchromatin is replicated 1st - Heterchromatin replicated last High repeat regions at centrosomes + telomeres also replicate last

Answer 95

lFlwo cytometry - cels from S phase fractions given BrdU (thymidine analogue) - anti-BrdU antibody with a fluorescent dye used to detect Immunopurified then sequenced to profile replication regions. If sequence replicated durig specific period - copy number will double in that sample ## Footnote Doesnt all happen at once, some early/late

Answer 96

When CDK + DDK high at onset of S-phase, initiation of replication can occur. Cdc45, Sld & GINS protein recruit replisome to initiate synthesis

Answer 97

Replication stress - causes DNA damage, potentially mutagenic RPA prevents DNA looping on itself by binding to exposed ssDNA PARP1 adds ribose residues, acts as tags for checkpoint kinase 1 (Chk1) to bind - activated Chk1 activates p53, S phase checkpoint activated - Cdk inhibtitors activated (p21, p27, Cdc25) - p21 + p27 block pregression to S-phase - Cdc25 blocks progression during S phase to allow repair (stalling mechanism) ## Footnote Repair proteins interact w/ stalled replisome at fork (PbR)

Answer 98

Dormant origins (backups) activated. - Mcm2-7 complexes displaced during initiation move along DNA to provide helicase function (unwind DNA) Beltand braces mechanism - reduces likelihood of errors ## Footnote MCM2-7 genes are prevalent in squamous cell carcinomas - when mutated, shown to causes cancer in mice.

Answer 99

mRNA that encodes histone normally very unstable Histone genes upregulated in S-phase (50-fold increase in mRNA) - timed by cell cycle

Answer 100

- H2A-H2B dimers released + held by histone chaperones, H3-H4 tetramers remain bound but rrandomly assigned to leading/lagging strand. - DNA Pol III duplicates each strand + chaperons randomly isnert H2A-H2B - new histone proteins intermix w/ parental histone (double toal mass) - histone methylase complexes recognise N-terminal methylation on parental + copy it to new histones locally (read write mechanism)

Answer 101

* Point mutations * Large scale INDELs * Chromosomal rearrangements

Answer 102

INDELs only one DNA base

Answer 103

Repetitive sequences, microsatellites - occur rapidly and can distinguish I dividuals based on number of copies.

Answer 104

Dramatic impact on organism function

Answer 105

* Inaccuracy in DNA replication * Damage to DNA

Answer 106

Biodiversity exists as a result of mutations

Answer 107

Single base pair change in DNA but no resulting amino acids change

Answer 108

DNA change results in changed amino acid sequence

Answer 109

DNA change encodes premature stop codon - almost always deleterious

Answer 110

1 mistake for every 10 copied base-pairs

Answer 111

* Proofreading * Mismatch repair

Answer 112

DNA Pol detects mistake via kink in DNA, slows down, exonuclease removes last added base Reduces error rate 100 fold

Answer 113

Corrects errors that escaped proofreading before permanent damage

Answer 114

Scans genome to find kinks and makes them more pronounced

Answer 115

Excises one strand near mismatch site

Answer 116

Unwinds DNA from the nick towards the mismatch

Answer 117

DNA Pol fills hole up with correct base-pairs

Answer 118

DNA ligase

Answer 119

methyl directed MMR MutH recognises strand to excise due to Dam methylase: - methylates DNA every 5'-GATC-3' on A, both strands - after replication, DNA hemi-methylated (only 1 strand carrying CH3) - MutH sits on methylated sites + nicks only uumethylated DNA (new strands) Distinguishes mutated vs original strands ## Footnote likely evolved form canonical repair system - MutL has function of both MutL & MutH

Answer 120

Inaccuracy in DNA replication, Damage to DNA, Spontaneous damage ## Footnote DNA damage can occur through various mechanisms, including errors during replication and external factors.

Answer 121

Occurs due to most chemical reactions in biology being reversible ## Footnote This means that the rates of reactions are not equal in both directions, leading to potential damage.

Answer 122

Deamination of cytosine makes uracil ## Footnote This process alters the base composition of DNA.

Answer 123

It makes apurinic deoxyribose, a sugar without a base ## Footnote This type of damage results in the loss of a purine base.

Answer 124

Thymine ## Footnote This process can lead to mutations in DNA.

Answer 125

Transfer of methyl/ethyl groups to reactive sites on bases or to phosphates in DNA backbone ## Footnote It can lead to wrong base pairing, such as thymine instead of cytosine in the case of 0-methylguanine.

Answer 126

Reactive oxygen species can cause mutations, such as OxG wrongly base pairing with adenine ## Footnote This is the most common mutation in cancer unless the organism is in an anaerobic environment.

Answer 127

260nm wavelength (UV radiation) ## Footnote It leads to the fusion of two pyrimidines, such as thymine dimers, preventing base pairing and stopping polymerase during replication.

Answer 128

Ames test ## Footnote This test assesses the mutagenic potential of chemical compounds.

Answer 129

Gamma radiation and X-rays ## Footnote These breaks are difficult to repair as there is no reference/template strand.

Answer 130

Prevents histidine synthesis ## Footnote This leads to a single point nonsense or frameshift mutation that converts his+ to his-.

Answer 131

[Alkylation]

Answer 132

Transfer of methyl/ethyl groups to reactive sites on bases or to phosphates in DNA backbone ## Footnote It can lead to wrong base pairing, such as thymine instead of cytosine in the case of 0-methylguanine.

Answer 133

Reactive oxygen species can cause mutations, such as OxG wrongly base pairing with adenine ## Footnote This is the most common mutation in cancer unless the organism is in an anaerobic environment.

Answer 134

260nm wavelength (UV radiation) ## Footnote It leads to the fusion of two pyrimidines, such as thymine dimers, preventing base pairing and stopping polymerase during replication.

Answer 135

Ames test ## Footnote This test assesses the mutagenic potential of chemical compounds.

Answer 136

Gamma radiation and X-rays ## Footnote These breaks are difficult to repair as there is no reference/template strand.

Answer 137

hisA gene in Salmonella knocked out so prevents histidine synthesis. - single point/nonsense converts his + to his- Reversal mutation in presence of mutagen converts his - back to his+ ## Footnote Grown on media w limited hisitidine Not a yes or no result - rough, indicative Variability between humans and Salmonella

Answer 138

Fastest changing DNA, not under stabilising selection so drifts and acquires mutations E.g. pseudogenes now don't have function but could in future

Answer 139

Direct reversal DNA damage Base excision repair Nucleotide excision repair Large scale damage repair

Answer 140

Removal of methyl group by methyltransferase -> O6 methylguanine (doesnt pair w/ C) Methyltrasnferase recognises DNA kink + uses exposed SH to bind methyl group -. removed from DNA

Answer 141

Photoreactivation - DNA photolyase detects dimers in dark, when exposed to light it induces removal of bond between thymine dimer (pyrimidine) -> free to base pair ## Footnote not 100% effective

Answer 142

Identifies damaged base pair (lesion) using glycosylase -> hydrolyses glycosidic bond leaving abasic sugar behind. Endo-exonuclease sutes 3' & 5' end removing abasic sugar + polymerase adds a new one. Ligase connects adjacent basic sugars

Answer 143

1 for each type of lesion: Uracil - damaged C 8-oxoG - damaged G 3-methyl adenine - damaged A

Answer 144

Does not recognise original strand so strugglr w/ mutation that has normal base but incorrect pairing (very rare) e.g. deamination of methylated C -> G:T pairing which is identified by TDG or Methyl-CpG Domain Protein 4 (MBD4) , converts T -> C

Answer 145

Recognises distortions, not lesions - caused by thiamine dimer or chemical adduct on a base Detection leads to removal of short ss segment, then filled w/ undamaged template strand

Answer 146

UvrAB complex scans DNA + finds distortion: - A detects then exits B melts DNA -> single stranded bubble around distortion, also recrutits UvrC

Answer 147

UvrC cuts DNA upstream + downstream of distortion, recurits UvrD UvrD (helicase II) releases single stranded garment from duplex DNA Pol synthesises new copy + liagse joins them tohether. ## Footnote Uvrs specific to E. coli but eukaryotes have similar systems - e.g. damaged DNA-binding protein (DDB) heterodimer of p120 & -48 subunits recognises specfic adducts

Answer 148

RNA Pol stalls when it reaches distortion - UvrA/B recruited, NER fixes damage allows RNA Pol to remain attached, transcription not compromised

Answer 149

Double stranded break repair In eukaryotes: homologous recombination In bacteria: Non-homologous end joining Translesions

Answer 150

No template so uses homologous recombination of chromatids. - ends resectioned by MRX comlex extended Exo1/DNA2 -> long 3' ssDNA strands sticky ends in damaged DNA - enables strand invasion so non damaged strands serve as remplate for damaged ones ( Rad51 forms a nucleoprotein filament on single-stranded DNA after end resection) - DNA pol extends damaged strand - Ligase joins strands -> Holiday junctions (4 stranded structure) - Holiday junctions resolved for repaired DNA ## Footnote only in eukaryotes as sister chromosome needed

Answer 151

Ligase glues 2 ends of of dsDNA break together - large permanent mistakes encoded in DNA but stabilises structure

Answer 152

Allows DNA Pol to cotinue replication despite disruption/damage. y-family polymerases recruited - incorporate new base pairs independently of parent base pairing. - replaced by DNA Pol III when beyond damaged section VERY high error rates - only want trasnlesion when under extreme stress ## Footnote machinery expressed as part of coordinated SOS response

Answer 153

Transfer of genetic material that is not 'vertical' - inherited. It can happen between any 2 organisms, most common in bacteria. ## Footnote Examples include bacteria exchanging plasmids, organelles transferring genes to eukaryotic chromosomes, and bacteria transferring genes to plants and insects.

Answer 154

Akiba and Ochia ## Footnote AR plasmids are circular DNA that carry genes conferring antibiotic resistance.

Answer 155

Agrobacterium causes crown galls, transferring bacterial causative genes into plant species for disease resistance. ## Footnote This process allows plants to gain useful traits from bacteria.

Answer 156

Gene HhMAN1 ## Footnote This gene allowed the beetle to digest coffee.

Answer 157

Survival in dark forests ## Footnote This was due to a gene transfer from hornwort, an aquatic plant.

Answer 158

It is a malaria-causing protozoa that can stay in the human body longer than other species. ## Footnote It acquired a block of genes from humans.

Answer 159

Uptake of free DNA from the environment. ## Footnote This can involve linear or plasmid DNA.

Answer 160

Linear DNA ## Footnote Linear DNA is more susceptible to damage due to open phosphate groups at the ends.

Answer 161

Circular DNA that requires cellular machinery to replicate. ## Footnote Plasmids can vary in size and copy numbers within a cell.

Answer 162

* Resistance * Virulence * Symbiosis ## Footnote These roles are important for the survival and adaptation of bacteria.

Answer 163

Griffith 1970s - S. pneumoniae R & S S is virulent + R is not (no polysaccharide capsule). But when R strain incubated w/ heat killed S strain -> mice died - tasnfer of DNA ## Footnote competence - some bacteri more transferable than others

Answer 164

Linear DNA degraded by nuclease -> ssDNA - ssDNA bound by competence-specific proteins that prveent its degradation - RecA incorporates ssDNA into chromosomes

Answer 165

Bacterial 'mating' - requires cell cell contact F plasmids in E.coli encode conjugation machinery - has tra apoeron, codes for sex pilli Pilli made by donor cell which find + attaches recipient(F- cell) + pulls it in (triggered by dircet cell contact, highly efficient) - DNA of donor plasmid nicked on 1 strand - Helicase spearates strands - DNA pol synthesises other strand when it has been transferred over via conjugation pilus

Answer 166

Large number - inherited by random diffusion, daughter cells likely to contain plasmid due to large quantity in parent Low number - random diffusion could skey plasmid inhertiance SO many plasmids code their own segregation machinery | can co-opt existing machinery by tying themselves to centromeres

Answer 167

Transfer of DNA through viral host - disocvered using p22 bacteriphage infection of salmonella Phage picks up host DNA + inadvertantly transfers to recipient - can pick up host DNA upon lysis of host cell Cannot always destroy host so transferred DNA integrated into host chromosome through lysogeny -> host genome divides, proliferating trasnferred DNA

Answer 168

Clover shape - has intramolecular base pairing 2 functional ends: - anticodon - 3'CCA end where a.acid binds, added by amino-acyl tRNA synthetase (uses ATP) Specific tRNA bases (dihydrouridine and inosine) affect base pairing + allow more complex tRNA secpndary structure.

Answer 169

Has 50S (contains PTC) & 30S (mRNA channel w/ decoding sites for binding tRNAs) subunits -> 70S 3 tRNA binding sites: - A (acceptor) binds aminoacyl-tRNA - P (peptidyl transfer) holds tRNA carrying nascent polypeptide chain - E (exit) where deacylated tRNA dissociates from ribosome Making ribosomes very costly (energy) - E.coli divides every 20 mins so must double protein count in this time ~60,000 ribosomes ## Footnote also has peotidyl trasnferase centre (PTC) & peptide exit tunnel

Answer 170

AUG - specifies start codon Met Shine-Delgarno sequence (AGGAGG) found 9+/-2 bases upstream of authentic start codon in bacterial mRNAs. Mirroed by anti-SD in 30S ribosome -> stabilises 30S-mRNA interaction ## Footnote SD interaction w/ anti-SD in 16S rRNA key for initiation site selection

Answer 171

Made of aptamer + expression platform: ligand binding eg metal ions, amino acids or nucleotides can block or reveal access to SD/ribosome binding site by stabilising mRNA secondary structure 'on switches' - expression correlates w/ ligand binding e.g. YkkC binds a guanidine activating expression of genes encoding export pump 'off switches' - ligand binding causes 'off' conformation e.g. AdoCbl riboswitches sense coenzyme B12 & switch off enzyme synthesis via feedback control to stop excess metabolite production

Answer 172

IFs guide Met-tRNA binding to AUG at P site in ribosome. - IF1 blocks A site to tRNA-met, inhibits premature 30s-50s interaction - IF2-GTP tags tRNA + regulates entry into ribosome - IF3 inhibits premature 30s-50s interaction, stabilises free 30s, accuracy check for tRNA-met binding

Answer 173

Ternary complex = aminoacyl tRNA-EFTu-GTP - binds A site + decodes - Peptide bond formed, peptidyl transfer of a.acid from P to A - Association of EF-G-GTP + ejection of empty tRNA from P site - Ribosome translocates, freeing up A site - peptidyl tRNA now in P site ## Footnote EF-Tu – mediates aminoacyl-tRNA entry to ribosome EF-G mediates translocation

Answer 174

- RF-GTP binds A site where termination codon appears - RF1 (UAG), RF2(UGA) & RF3-GTP hydrolyse polypeptide chain from tRNA - tRNA & RF dissociate

Answer 175

Prevent translation or lter its fidelity by targeting ribosomal functional centres 30S antibiotics target decoding step in elongation (prevent it or allow errors) - e.g. tetracycline, neonmycin, streptomycin 50S antibiotics interfere w/ peptide bond formation (via PTC) e.g. streptogramin A/B, chloramphenicol ## Footnote ^bacteriostatic

Answer 176

Mutations prevent antibiotic binding -> can give reistance to other antibitoics as there is significant overlap in binding sites ## Footnote VRSA (S. aureus) caused by mutation in vanA gene, trasnposon or HGF.

Answer 177

- mRNAs have single ORF , 5' cap + polyA tail vs prokaryotic mRNAs often in operons - nuclear membrane w/ splicing - transcription + translation separated vs prokaryotes it is coupled. So ribosomes recruited to 5' cap & then scan to locate Kozak sequence + AUG start codon ## Footnote 5' & 3' UTRs vital for translation control

Answer 178

Initiator tRNA binds 40S subunit, ribosome binds near 5' cap, but in wrong place. 43S PIC binds mRNA -> 48S, ribosome moves along sequence scanning to find AUG (1st one found is used unless it has poor kozak sequence) Large 60S subunit joins

Answer 179

- disassembled 40S subunit needs to bind eIF1, 1A & 3 - eIF2 binds met-tRNAi when bound to GTP -> ternary complex (TC) - both combine w/ eIF5 to form 43S PIC Initiator met-tRNAi binds 40S ribosome in complex w/ eIF2 (+1, 1A, 3 & 5)

Answer 180

mRNAs primed by eIF4E binding at 5' cap w/ eIF4G & poly-A binding protein (PABp) -> mRNAs circularise forming closed loop complex eIF4A/B unwinds any mRNA secondary structure -> landing site for 43S complex near 5' end 43S lands so met-tRNAi & mRNA can base pair - codon/anitcodon interaction can be foun during scanning

Answer 181

AUG codon-anticodon pairing triggers eIF1 release + eIF2-GTP hydrolysis (does not bind met-tRNAi so released) eIF5B-GTP binds, promoting 60S association + other factors released

Answer 182

eEF1A-GTP: binds all tRNAs except initiator to A site (EF-Tu), activated by eEF1B (GDP/GTP exchanger, EF-Ts) eEF2-GTP: mediates translocation (EF-G) eEF5A: assists in peotide bond formation (EF-P)

Answer 183

eRF1: recognises all 3 stop codons in A site + catalyses release of nascent polypeptide from tRNA in P site eRF3-GTP: regulates ribosome binding via GTP hydrolysis

Answer 184

- increases speed of effect on protein levels - allows local control in specific part of cell Translation often inhibited transiently while stress is resolved. Can be activated -> faster growth, upon stimulation by hormones, GFs, mitogens + cytokines - globally, during development - locally, wound healing

Answer 185

- synergistically enhance translation of all eukarytic mRNAs Conducted experiment w/ mutants of both elements- luciferase used as reporter gene (fluorescence). - found to be vital for translation, RLU decreased in mutants. bind eIFs to direct ribosome entry & binding

Answer 186

Polysome profiling - inidcates levels of 40S, 60S, 80s + polyribosomes. Cells treated with cycloheximide to freeze all ribosomes at current positions on mRNA. - cell extracts prepared + centrifuged Can compare under differetn conditions. e.g. increasing H2O2 inhibits protein synthesis due to oxidative stress - new proteins can also be labelled w/ 35^S methionine

Answer 187

Phosphorylation of eIF2 at a-serine 51by Gcn2 inhibits eIF2B GEF activity. -> translation attenuated as ternary complex not formed Transient stress repsonse is good, but prolonged is bad (see footnote) ## Footnote aberrantly high eIF2A phosphorylation by PKR kinase -> seen in brain w/ Down Syndrome, protein synthesis needed for LTP + synaptic plasticity (associated w/ memory)

Answer 188

eIF4E binding proteins inhibit translation - prevent 4E intract w/ 4G - mRNA recuritment blocked so translation shut down 4E-BPs & eIF4g have common sequence motif -> YXXXXL(delta), binds same part of eIF4E

Answer 189

mTOR (kinase) promotes growth & translation by inactivating 4E-BPs via phosphorylation. 5'TOP mRNAs that have 5' terminal oligopyrimidine run (C/U) are most affected by 4E-BP1/2 mTOR control. - found in ribosomal proteins + TF mRNAs, so control tranlation of everything else Thr36 + 45 are key phos sites -> dramatic folding from a-helix to B-strand - new folding buries 4E-BP Y53 in B-strand so lowers affinity for eIF4E >1000 fold eIF4E free for translation | 5'TOP - 5' terminal oligopyrimidine ## Footnote adding 4E-BP1 slows translation, slowing tumour growth in breast cancer cells

Answer 190

Work w/ global controls -> precise regulation of transaltion specific proteins. Features for controls: - 5'cap & polyA tail promote translation on all mRNAs - 5' & 3' UTR critical for controlling translation 1) Ferritin mRNA 2) Arg-2 mRNA 3) Nanos mRNA

Answer 191

Iron levels in mammals - aconitase/IRP1 regulates ferritin mRNA translation. High [Fe] - aconitase is a 4Fe-4S cluster binding enzyme, converts citrate -> isocitrate in TCA cycle - RNA helicase (eIF4A) allows 40S scan to find ORF AUG Low [Fe] - iron limiting, aconitase inactivated, transforms into IRP1 -> binds stem loop structure (IRE) in ferritin mRNA 5'UTR - prevents 40S access + scanning

Answer 192

Arg levels in neurospora. R biosynthesis regulated by both R levels & a uORF encoding AAP - Arg-2 makes R when R levels low, repressed by high R Control requires: - normal scanning - leaky scanning of AAP uORF to translate Arg-2 - ribosome & AAP sequence to monitor excess Arg Low Arg - leaky scanning of uORF (only 50% ribosomes translate AAP, poor kozak sequence), so ARG-2 translated, those that translate AAP dissociate from mRNA High Arg - excess Arg stalls ribosomes translating AAP at stop codon, Arg binds AAP peptide in exit tunnel blocking AAP release - stalled ribosomes block further ribosome movement - Arg-2 not translated | AAP - Arg attenuator peptide ## Footnote Principle: uORF in 5'UTR can control ribosome access to a main ORF + bring about condition-specific regulation, ~50% human mRNAs have uORFs.

Answer 193

Early embryonic development in fruit fly. A-P axis must be established - requires post-transcriptional control of maternal mRNAs. Cup protein is 4E-BP that regulates translation + location of Nanos protein. Nanos repressed everywhere except posterior end - directed by mRNA 3'UTR binding proteins (Smaug & Glo) - interact w/ stem loop RNa structure in 3'UTR - form closed loop w/ Cup so eIF4G cannot access mRNA * ensures only specifc mRNas transaltionally repressed -> localisation in a cell + polarity Nanos is a translational regulator of hunchback ## Footnote Principle: 4E-BP tethered to sequence or structure in 3'UTR via specific RNA-binding proteins can control ribosome access -> spatially regulated translation - Also happens in mammals inc. neurons to regulate memory

Answer 194

Antizyme mRNA - promotes programmed frameshifting in mammalian cells Common in viruses (HIV, SARS-Cov2) - antigenic drift. In eukaryotes: +1 frameshift controls polyamine levels in cells (spermine, spermidine, putrescine, ornithine) AZ levels controls ODC levels -> tight control of polyamine levels. - delivers ODC to proteasome for degradation + inhibits polyamine uptake mRNa has 0 frame (ORF1) w/ stop codon + +1 frame w/ no start codon SO ribosome translates ORF1 OR longer ORF1-ORF2 (which is functional antizyme enzyme) 3' pseudoknot is modified stem loop, stalls ribosome so it can attach 2nd reading frame (has shifty segment) | ODC - ornithine decarboxylase enzyme ## Footnote Excess spermidine promotes ribosome frameshifting feed back control by binding ribosome. Principle: elongation + reading frame maintenance can be affected by combination of local sequences + small molecules binding to ribosome.

Answer 195

- 5' end capping (GTP binds 5' end + 2' pos of 1 st 2 nucleotides methylated) - intron splicing: signalled by conserved sequences + branchpoint region (spliceosome is complex of snRNPs) -> U2 + U5 + U6 make up active site - 3' end cleavage + polyadenylation (CPSF binds AAUAA, CstF binds G/U -> Poly-A pOlymerase + cleavage factors recruited) + mRNA specific events: RNA editing, alternative splicing

Answer 196

7-methylguanosine connected to 5' end via triphosphate bridge. Recognised in nucleus by CBP20/CBP80 -> stability, connection to export apparatus + connection to splicing (lariat formation) In cytoplasm: interacts w/ eIF4E -> RNA stability, nuclear export + translation initiaiton ## Footnote its location dictated by site of transcription - regulated by alterantive promoters

Answer 197

1993 Roberts + Sharp shared Nobel prize - hybridized denatured adenoviral DNA fragment to purified exon mRNA Conserved consensu sequences define splice site. e.g. Branchpoint (YNYURAC) is 30 bases upstream of 3' end - connected by polypyrimidine tract 1) 5' splice site binds to 2'-OH at branchpoint forming loop (spliced from exon 1) 2) exon 1 joins to exon 2 via 3' splice site -> phosphodester linkage (upstream introns spliced out) U1, 2, 4, 5 + 6 snRNAs very important - U1 frees 5' end so it can base pair to specific sequences -> snRNPs precisely target intron/exon boundaries | Y - pyrimidine (C & U) R - purine (A & G) N - any

Answer 198

- Commitment/E complex formed by SR-U2AF interaction - U2 then recruited SR associates w/ RNA Pol II tethering transcription to splicing

Answer 199

Exons of primary transcript reconnected in various ways -> altered protein products (isoforms) - relies on efficieny of splice signals (donor/acceptor) - extra enhancer/silencer sequences control this efficiency SR proteins bind splicing enhancers dictating where it happens hnRNP protein (hnRNPA1) binds silencing enhancers ## Footnote many different effects on protein

Answer 200

Dscam1 mRNA processing Every neuron in fruit-fly expresses different version on its surface. - tiny variations in specificity + function between isoforms -> 38,016 different mRNAs from 1 primary transcript

Answer 201

5' -> 3': deadenylation , decapping & 5'->3' exonuclease activity 3' -> 5' exosome: deadenylated mRNas degraded by exosome ^ redundancy in both pathways- can pick up each other slack

Answer 202

- pop2/Ccr4 complex -> deadenylation - Lsm1-7 binds polyA after PABP removal + recruits decapping proteins - Dcp1/2 carry out decapping of 5' end. - Xrn1 nuclease cleaves mRNA ## Footnote PABP - polyA binding proteins Lsm1-7 is a marker protein complex -> necessary to recruit decapping proteins.

Answer 203

- pop2/Ccr4 complex -> deadenylation - Exosome/Ski complex binds 3' end -> 9 subunits w/ ring shape -> has 3'->5' exonucleases + other RNases, RNA helicases, porcess other RNAs - Dcp1/2 removes 5' end left over (scavenger decapping enzyme)

Answer 204

Cytoplasmic bodies that contain: - components to 5'->3' decay pathway - components to RNAi pathways Possibly store mRNA for future degradation or use. ## Footnote mRNA scrapyard

Answer 205

Cap binding complex inhibits acces of deadenylase + decapping enzymes. - inhibition of initiation will promote mRNA decay Aberrant mRNAs are degraded via specialised mRNA surveillance pathways: - NMD - NSD - NGD

Answer 206

Identifies mRNAs w/ premature stop codon. - either due to mutation/errors or incomplete splicing EJC & CBP20/80 recruits 3 UBF proteins to where ribosome stalled -> UBF1 phosphorylated & mRNA endonucleolytically cleaved | Exon junction complex (EJC) is a marker for where splicing has occurred

Answer 207

Identifies mRNA with no STOP codons - either stop codon mutated or premature polyadenylation occurs Lack of stop codon means ribosome displaces PABP (acts as buffer) - UPF recruited, then exosome for 3'->5' decay

Answer 208

Deals with issues during translation. If ribosome stalled due to secondary structure - causes decay. Dom34/Hbs1 complex recruited to stalled ribosome - dissociates ribosome + prootes endonuclease cleavage. 5' end mainly degraded by 3'->5' but both pathways used (redundancy)

Answer 209

Regulates amount of mRNA made. 3'UTR examples include: 1) regulation of mRNA stability by AREs 2) Transferrin receptor mRNA & IRP1 (IREBP1) | ARE - AU rich element

Answer 210

i- usually destabilisng but can be bound by proteins which stabilise it (impacts onTFs, cell cycle, cytokine/lymphokines) -> proteins include: AUF1 (destabilisation of c-fos, c-myc, IL3) , HuR (stabilises AU elements in Cyclin A1/D1) & TTP Destabilisation - RBP recruited + interacts w/ decapping proteins + deadenylation complex

Answer 211

- IRP1 binds stem loops in Tfr 3'UTR + blocks access of ARE-BPs -> stabilising mRNA - Fe binding to IRP1 causes its dissociation from AU rich sequences -> ARE-BPs can access mRNA + induce degradation ## Footnote self regulating mechanism - swithes off when plent of Fe in cell

Answer 212

Post-transcriptional gene silecnign mediated by short RNA mols i.e. miRNA/siRNA Initiation step - siRNAs generated Effector step - degradation of target mRNA

Answer 213

Microprocessor converts pre-miRNA into ds-stem loop structure. - dsRNA cleaved by dicer + phosphorylated by putative kinase - RdRp amplifies siRNA (not in mammals) ## Footnote Pathway above generalised - actually species specific

Answer 214

Atp-dependent helicase w/ dsRNA binding domain & RNase III activity. - only 1 in C. elegans/mammals but > 1 in Arabidopsis/Drosophila Work as dimers to generate small 22-24 bp siRNAs

Answer 215

siRNA binds mRNA by base pairing + unwinds RISC activated to: - inhibit translation in animals - degrade mRNa/ silence heterochromatin in plants | RISC - RNA-induced silencing complex

Answer 216

- 2 RNA binding proteins - RNA/DNA helicase - translation initiation factor - RdRp in some species where effct needs to be amplified - TM protein -> propagates initiation across cell pop. RISC-siRNA -> mRNA degradation miRNP-miRNA -> tranlsation initiation RITS-rasiRNA -> heterochromatin modification ## Footnote All have argonaute proteins (indicative)

Answer 217

RNA that doen not encode protein but may stoll contain info or have function. Encoded by RNA Pol II: short - snRNA, snoRNA, miRNAs, siRNAs long - lincRNAs, eRNAs, PROMPTs rRNA & tRNA encoded by RNA Pol I & III

Answer 218

Dependent so co-expressed w/ mRNA Inc promoter associated (paRNA) or transcription start site (TSS-ncRNA). Transcribed in same direction as sense mRNA, 20-60nt long + non-functional - BUT important check in point in gene expression regulation

Answer 219

Trabscribed in revrse orientation to sense mRNA -eukaryotic promoters often bidirectional so trancription can initiated both directions. 0.5-1.5kb long, non-functional If ncRNAs not terminated - transcription would evetually be blocked by Pol transcribing PROMPTs - uncontrolled pervasive transcription -> lethal interference Pol II blocking each other -> tension -> DNA breaks

Answer 220

Integrators + restrictors used. Transcription of ncRNAs can be initiated anywhere inc terminators Non-productive veents immediately restricted to avoid interference

Answer 221

small nucleolar RNAs (snoRNAs) are embedded in introns. Co-assmebled during mRNA splicing - trimming takes place to form mature snoRNP from an intron lariat. - uses 5' and 3' degradation Functions: - RNA methylation (box C/D) - RNA pseudouridinylation (box H/ACA) they recognise RNA targets via short complementary sequences ## Footnote mainly independent in lower hosts

Answer 222

Prader-Willi caused by large DEL in chrom 15 - snoRNa cluster involved, some can form long non-coding RNAs (sno-lncRNAs) Requires 2 sno-RNA in same intron SO exonuclease cannot degrade -> very stable mol w/ sno-RNA at both ends. ## Footnote Sno-lncRNAs one of most abundant RNA species in human stem cells

Answer 223

Expressed independently of protein-coding genes - transcribed from own promoters small nuclear RNAs (snRNAs) - in all eukaryotes (pre-mRNA splicing) - for riboprotein complexes (U1, 2, 4, 5, 6) - short + fold into structural RNAs - terminated by integrator as non-polyadenylated ^ all transcribed by Pol II except U6 (Pol III) Enahncer RNAs (eRNAs) also independent - cis-regulatory elements, cooperate w/ promoters to regulate expression - highy transcribed - terminates by either cleavage & polyadenylation , restrictor/integrators complexes

Answer 224

Non-polyadenylated - bidirectionally transcribed so terminated by restrictor/integrator complexes Polyadenylated - unidirectionally transcribed (>4kb), trasncirbed by v. active enhancers, terminated by cleave + p. adenylation Regulation of expression (putative): - molecular glue - sequestering - phase separated environemnt

Answer 225

lincRNAs - independent transcription units - many function + tissue specific BUT synthesis more inefficient than mRNA + mainly in nucleus Mostly associated w/ higher order structure + binding proteins. - associated w/ many different diseases ## Footnote lincRNAs are 'non-coding copies' of mRNAs: capped, spliced & polyadenylated. lincRNAs usually localised in the nucleus + many lincRNAs are functional

Answer 226

DNA sequences that have ability to change position within a genome. Either replicate sequence + insert into another gene (copy + paste) OR sequences removed from one locus + moved to another (cut + paste) ## Footnote 46% human genome

Answer 227

Barbara McClintock studied mechanism of unstable inheritance of mosaic colour patterns in maize seeds. Described loci disscoiation (Ds) & activator (Ac) - both could change genome. Progenitors of TEs likely present in common ancestor of all eukaryotes - originates from viruses or actively transcribed small RNA genes

Answer 228

DNA transposons - mobilised via DNa intermediate - cut & paste mechanism - flanking direct repeats play role in TE insertions (left behind as footprints after TE excised) Inverted terminal repeats (ITRs) complementary to each + recognise transposase

Answer 229

Retrotransposons rely on RNA Pol & reverse transcriptase - RNA interemdiates & copy-paste mechanisms Subclasses are long terminal repeats (LTRs) & non-LTRs (LINE & SINE)

Answer 230

Presence of LTRs, produces target site duplications (TSDs) upon insertion. ORF encodes rev. transcriptase, ribonuclease H + integrase -> enzymatic machinery for integration

Answer 231

long interspersed elements: - pol II promoter - ORF1 encodes RNA binding chaperone - ORF2 encodes endonuclease -> ss nick in DNA, rev. trasncriptase uses nicked DNA to prime rev. transcription - polyadenylation site followed by A(n) ## Footnote non-LTR subclass

Answer 232

short interspersed elements: - do not encode any of own retrotranscription machinery, mobilised by L1 machinery - contains components of RNA Pol III promoter - Alu SINEs contain Alu restriction site

Answer 233

- If strongly deleterious, rapidly removed - If little/no effect then any reach fixation rarely fixed in exons & depleted from regions coding ncRNAs

Answer 234

Non allelic homologous recombination (NAHR) e.g. MLL-1 & BRCA1 hotspots fror Alu-Alu recombination -> gene duplications, rearrangements + deletions BRCA genes TE insertion -> breast cancer

Answer 235

Especially in eukaryotes - limited HGT like in bacteria. - Can shuffle exons or even become them. - Faciltate translocation of genomic sequences e.g. - Rag1 & Rag2 catalyse sonatic recombination in vertebrates - Gag-derived Arc gene involved in memory + synaptic plasticity ## Footnote Gag polyprotein integral to viral structures

Answer 236

Human cells employ chromatin remodelling. - HUSH & KRAB-ZNF regulates methylation of Lys9 (H3K9me3) - Polycomb complex mediates H3K27me3 -> heterochromatin formation to keep TEs inactive

Answer 237

Small + compact, genes relocated to nucleus -> better control mechanisms Peptide sequences have targeting signal -> directs proteins back to organelle

Answer 238

- maternally inherited - different structure + code to nuclear genome Bidirectional replication by DNA Pol gamma, leading strand synthesis starts at specific open D-loop Human mtDNA is circular loop - transcribed from both strands as long polycistronic precursor - pre-mRNAs processed by tRNA punctuation model

Answer 239

tRNAs act as punctuation marks for mtRNAs -> excised by endonucleases (RNase P/Zs, Z cleaves 3' end, P cleaves 5' end) 22 interspersed tRNAs removed to release rRNAs + mRNas ## Footnote In higher eukaryotes, RNase P encoded in nucleus then imported into mt

Answer 240

Has its own ribosomes. In mt mRNA: - no cap - no to be spliced - polyadenylated after transcription by mtPAP shorter polyA tail in mtRNA

Answer 241

In mt, chloroplasts + bacteria, have exonucleolytic + polyadenylation activity. Human PNPase is exonuclease only -> degrades RNA ## Footnote ATP dependent hSUV3 unwinds secondary structures

Answer 242

MELAS MERRF LHON ## Footnote mtDNA mutation rate ~30 fold higher than

Answer 243

Same cell can tolerate varying proportions of mutated & normal mtDNA

Answer 244

Heteroplasmy shifting - targeted removal of mutated mtDNA OLD: mitochondrially targeted restriction endonucleases NEW: programmable nucleases w/ sequences specific DNA binding domains: - zinc finger nucleases (ZFNs) - transcription activator like effector nucleases (TALENS) ^ linked to seuqnece independent endonuclease (Fokl)

Answer 245

- maternally inherited, codes for proteins in photoysnthesis - has same genetic code as nuclear genome 2 origins from which elongation of nascent strands initiated - fork movements towards each other + fusion of D loops (bidirectional) highly conserved genome, excised by exo- and endonucleases

Answer 246

- no cap - may have introns - polyadenylation marks for degradation