Biol 200 Final

Front 1

Nucleosides

Back 1

-just the nitrogenous base no phosphate groups

Front 2

Amino Acid Isomers

Back 2

-D and L

-humans have adapted to use L isomers

Front 3

Cysteine

Back 3

-can form disulfide bonds with another cysteine between sulfhydryl groups

-stabilizes folding structure

Front 4

Glycine

Back 4

-small and compact due to symmetrical hydrogens

Front 5

Proline

Back 5

-rigid ring structure

Front 6

Coiled-coil motif

Back 6

-derived from fibrous proteinso

Front 7

Motifs and Domains

Back 7

motifs: combinations of secondary structures

domains: large stretches of amino acids that fold to give rise to functional regions of proteins

Front 8

EF hand/helix-loop-helix motif

Back 8

-ionic bonds involving proteins and Ca2+

Front 9

Zinc-finger Motif

Back 9

-series of histamines/cysteines

Front 10

B form of DNA

Back 10

-most commonly found in cells

-major and minor grooves that allow DNA binding proteins to interact w/ double helix

Front 11

A DNA

Back 11

-occurs in low humidity/dehydrated samples of B DNA

-shorter and more compact

-RNA-DNA and RNA-RNA helices exist in A form

Front 12

Z DNA

Back 12

-short DNA molecules that adopt left handed helix configuration

-transiently formed after transcription

-has been found in cells

Front 13

Holoenzyme

Back 13

-two alpha and two beta subunits plus a sigma factor

Front 14

Sigma Factor

Back 14

-essential role in selecting site of transcription initiation

-finds promoter

Front 15

Aminoacyl-tRNA synthetase

Back 15

-recognize and conjugate specific amino acids

-also recognize and bind to cognate tRNAs

-links amino acids to corresponding tRNAs

-uses ATP to generate high energy ester bonds between the 3' end of the tRNA and amino acid (aminoacyl-tRNA)

Front 16

Initiation Factor 1

Back 16

-associates with 30S subunit

-assists with loading preinitiation complex onto mRNA

-later recruits the 50S ribosomal subunit

Front 17

Initiation Factor 3

Back 17

-associates with 30S subunit

-assists loading of preinitiation complex onto mRNA

Front 18

Initiation Factor 2-GTP

Back 18

-recruits 50S ribosomal subunit to form 70S initiation complex

Front 19

23S rRNA

Back 19

-ribozyme

-carries out peptidyltransferase reaction during elongation of peptide chain

Front 20

Shine-Delgarno box

Back 20

-5-8bp sequence that can bind to 16S rRNA

-just before AUG sequence

-required for bacterial translation initiation

Front 21

Release Factors 1 and 2

Back 21

-mimic tRNAs

Front 22

eIF2-GTP

Back 22

-initiation factor in eukaryotic translation

-forms ternary complex with Met-charged tRNA

-can engage in 40S complex with other initiation factors

-can negatively regulate protein synthesis through phosphorylation

-hydrolysis to eIF2-GDP forms 48S initiation complex

Front 23

eIF5

Back 23

-eukaryotic initiation factor

-joins 40S complex to form 43S pre-initiation complex

Front 24

Dimeric Guanylyltransferase

Back 24

-binds to phosphorylated CTD of RNA Pol II after mRNA transcripts are capped

Front 25

eIF4

Back 25

-eukaryotic initiation factors in translation

-efficiently binds to 5' cap on mRNA

-recruits mRNA to pre-initiation complex

-eIF4E binds to cap

-eIFG binds to eIF4E and PABP1 to form loops that allow for easy re-initiation of translation

-eIF4A binds to RNA helicase

Front 26

KOZAK sequence

Back 26

-ACCAUGG

-in mammalian translation

-relatively conserved (mainly A and G)

Front 27

eIF1A

Back 27

-binds to GTP

-forms complex with tRNA to interact with A site

-GTP is hydroylzed if tRNA anticodon matches the codon

-eIF1a-GDP leaves after hydrolysis

Front 28

Elongation Factor 2

Back 28

-bound to GTP

-used to move ribosome forward to translocate tRNAs into E and P sites

Front 29

eRF1

Back 29

-termination release factors in eukaryotes

-mimics aminoacyl-tRNAs

Front 30

eRF3-GTP

Back 30

-undergoes GTP hydrolysis to catalyze the cleavage of peptidyl-tRNA

-associated with eRF1 bound to A site

Front 31

ABCE1 ATPase

Back 31

-dissociates post-termination complex

Front 32

MSH1/MSH6

Back 32

-proteins that bind to daughter strand with incorporated error during mismatch excision repair

Front 33

Large T-antigen

Back 33

-encoded by SV40 virus

-hexamer

-opens DNA at high rates (helicase)

Front 34

Replication Protein A

Back 34

-bind to single stranded DNA

-keeps template in optimal configuration for DNA Pol A

Front 35

Origin of Replication Complex

Back 35

-six subunit protein

-binds to replication origins

-associates w/ proteins to load helicases

Front 36

PCNA

Back 36

-proliferating cell nuclear antigen

-homotrimetric protein

-binds around polymerase

-prevents Pol/Rfc/PCNA complex from dissociating from template

Front 37

Topoisomerase

Back 37

-relieves supercoils in DNA

Front 38

Primase

Back 38

-synthesizes short RNA sequences called primers

Front 39

DNA Polymerase Alpha

Back 39

-recognizes primer area well

-not very good at extending

-initially required for leading and lagging synthesis

Front 40

DNA Polymerase Epsilon

Back 40

-required for full extension of DNA leading strand

-proofreads errors made in base pairing with Polymerase Delta

Front 41

DNA Polymerase Delta

Back 41

-fully extends DNA lagging strand

-replaces gaps left by RNA component removed by ribonuclease H/FEN-1

-participates in proofreading with Pol E

Front 42

DNA Polymerase Beta

Back 42

-participates in base excision repair

-fills gaps left by removed incorrect base

Front 43

Ribonuclease H/FEN-1

Back 43

-displace RNA component of 5' ends of Okazaki fragments

Front 44

DNA glycosylase

Back 44

-participates in base excision repair

-hydrolyzes bond between mispaired base and sugar phosphate backbone

Front 45

APE1

Back 45

-cuts sugar phosphate backbone in base excision repair

-endonuclease

Front 46

AP lyase

Back 46

-endonuclease

-associated with Pol Beta it removes part of sugar phosphate backbone during excision repair

Front 47

MLH1

Back 47

-endonuclease

-cuts sugar backbone during mismatch excision repair

Front 48

TFIIH

Back 48

-joins open core pre-initiation complex to open it before transcription

-only general/basal transcription factor that has ATP dependent enzymatic activities

-acts as helicase with XP-G to open DNA during nucleotide excision repair

-has carboxy-kinase needed to phosphorylate CTD

-using ATP, helicases in TFIIH melt promoter around transcriptional start site (transcription bubble)

Front 49

MLH1 Endonuclease

Back 49

-participates in mismatch excision repair

Front 50

Protein 23B

Back 50

-associates with XP-C to recognize thymine dimers in nucleotide excision repair

-allows for opening of helix

Front 51

XP-C

Back 51

-protein complex

-scans DNA for distortions

-recognizes kink in DNA caused by thymine dimers in nucleotide excision repair

Front 52

XP-G

Back 52

-associates w/ TFIID to open DNA helix during nucleotide excision repair (helicase)

-also cuts out section of single stranded DNA during nucleotide excision repair (endonuclease)

Front 53

XP-F

Back 53

-acts as endonuclease in nucleotide excision repair

-cuts out section of single stranded DNA during with XP-G

Front 54

DNA-PK

Back 54

-DNA dependent protein kinase

-recognizes breaks in DNA strand during end-joining

Front 55

Ku80/Ku70

Back 55

-heterodimer

-binds to ends of break in DNA strand during endjoining

Front 56

RecA

Back 56

-recognizes 3' overhangs during homologous recombination

-facilitates strand invasion

-in prokaryotes

Front 57

Rad51

Back 57

-recognizes 3' overhangs during homologous recbomination

-facilitates strand invasion

-in eukaryotes

Front 58

Plasmids

Back 58

-most common vector used in recombinant DNA technology

-circular double stranded DNA found in bacteria/lower eukaryotes

-extrachromosomal

Front 59

Polylinker

Back 59

-site on plasmid vector consisting of variety of recognition sequences for restriction enzymes

Front 60

ddNTPs

Back 60

-terminal nucleotides, dideoxynucleotides

-no hydroxyl group on 3' carbon of sugar

-utilized in DNA sequencing (Sanger, automated, etc)

Front 61

Transposons

Back 61

-mobile gene elements found in prokaryotes and eukaryotes

-selfish

-make up large portion of human genome (mostly non functional)

Front 62

DNA Transposons

Back 62

-"cut and paste" mechanism

-autonomous (have transposase gene) and non autonomous

-special flanking sequences: target site direct repeats and target inverted repeats

Front 63

Retrotransposons

Back 63

-act like retroviruses (act through RNA intermediate)

-encode for reverse transcriptase

Front 64

LTR Retrotransposons

Back 64

-6-11kb

-target site direct repeats (5-10bp)

-Long Terminal Repeats (250-600bp)

-ORF order: gag, pol, env (non functional)

Front 65

Gag

Back 65

-polyprotein

-encoded by retroviral genome

-group specific antigen

Front 66

Pol

Back 66

-encoded by retroviral genome

-reverse transcriptase function

Front 67

Env

Back 67

-encoded by retroviral genome

-envelope protein

-allows retrovirus to leave host cell

Front 68

LINEs

Back 68

-Long Interspersed Elements

-no LTRs

-6-8kb in length

-present in L1

-ORF1 and ORF2

-autonomous

Front 69

-ORF1

Back 69

-Open Reading Frame 1

-on LINEs

-codes for RNA-binding protein

Front 70

ORF2

Back 70

-Open Reading Frame 2

-on LINEs

-codes for reverse transcriptase and DNA endonuclease

Front 71

SINEs

Back 71

-short interspersed elements

-mobilized non-coding RNA genes

-100-400 bp in length

-most abundant type of mobile element in human genome

-tRNAs are SINEs

-non autonomous

Front 72

Processed Pseudogenes

Back 72

-mobilized protein-coding genes

-originate from mRNA that was accidentally reverse transcribed and inserted into genome

-lack introns and control regions

-often not expressed

Front 73

RAG1/RAG2

Back 73

-"domesticated transposon" gene

-derived from transposase gene

-encodes for recombinases important for assembly of human immunoglobin genes

Front 74

Syncytin

Back 74

-"domesticated transposon" gene

-encodes for DNA binding proteins that mediate placental cell fusion

-derived from LTR-retrotransposons

Front 75

Insertion Mutagenesis

Back 75

-transposon lands in gene

-causes gene to become non-functional

-ex. maize , snapdragon, wine grape colours

Front 76

Gene and Segmental Duplication

Back 76

-gene duplication results from uneven cross over

-ex. alpha/beta hemoglobin genes

Front 77

Exon Shuffling

Back 77

-mediated by recombination between mobile elements

-double cross over between mobile elements

-transposons sometimes bring exon between them into another gene

-LINE segments sometimes have weak Poly A signals

Front 78

Cis-regulatory Module

Back 78

-region of DNA where proteins bind and regulate expression of nearby genes

-controls temporal/spatial regulation of gene

-controls how much mRNA transcript will be produced from gene

-ex. of diversification includes insecticide resistance, domestication of corn and dogs

-over evolution, selection will only be on maintenance of CRM and not rest of transposon

Front 79

Transduction

Back 79

-processed host gene within LTR-retrotransposon is reverse transcribed and inserted into host genome

Front 80

Transduplication

Back 80

-host genomic region within a DNA transposon

Front 81

Organellar DNAs

Back 81

-ex. mitochondria and chloroplasts

-originally free organisms that were endocytosed and became endosymbionts

-resemble prokaryote genomes (circular, lacking introns, produce gene products resembling prokaryotic RNAs/proteins)

Front 82

Mitochondria DNA

Back 82

-16 569 bp (humans)

-37 genes

-no introns

-gene products remain within mitochondria

-mutations in mtDNA may be related to aging in mammals

-UGA stop codon read as trp

-maternally inherited

-multiple copies of mtDNA per mitochondria

-greater divergence in mtDNA than in nuclear DNA

Front 83

DNA Barcoding

Back 83

-sequence and identify species/differentiate

-PCR strategy to find sequence

-primers flank "barcode sequence"

-barcode selection:

-divergence (not to high or low)

-must be able to be amplified by PCR (short, flanked by conserved region)

-must be easily aligned (no insertions/deletions)

-BOLD database

Front 84

Chromatin

Back 84

-nucleo-protein complex

-when cells are not dividing DNA is found in chromatin

-extended and compact forms

Front 85

"Beads on a String"

Back 85

-extended form of chromatin

-bead = nucleosome

-string = 10-90bp of linker DNA

Front 86

Solenoid Model of 30nm fiber

Back 86

-compact form of chromatin

-6 nucleosomes and linker DNA aligned in circle

-forms spiral with 30nm diameter

-inner ring of H1 histone

-most widely accepted model of condensed structure

Front 87

Two start Helix model of 30nm fibre

Back 87

-nucleosomes form stack of "coins"

-stacks form helices

Front 88

Nucleosomes

Back 88

-histones and DNA

-consist of disc-like protein core (H2A, H2B, H3 and H4) with DNA (147 bp ) wound around surface

-DNA has to be removed from histones during cell replication

Front 89

Histones

Back 89

-highly alkaline proteins that make up protein core of nucleosomes

-5 major types

-H1, H2A, H2B H3 and H4

-rich in postively charged amino acids (facilitates interaction w/ negatively charged phosphate groups of DNA)

-have flexible end terminal sequences not bound to DNA

-required for chromatin condensation

-modification of histone tails regulate chromatin condensation (ex. acetylation of lysine neutralizes positive charge)

Front 90

H2A

Back 90

-histone type

-highly conserved among distantly related species

-has C-terminal tails

Front 91

H2B

Back 91

-histone type

-highly conserved

-N-terminal tails

Front 92

H1

Back 92

-histone type

-variable

Front 93

Histone Code

Back 93

-particular combinations of modifications collectively found in chromatin

-can create/remove chromatin-protein binding sites

-determine which regions of genes in chromatin are transcribed

Front 94

SARs/MARs

Back 94

-specific DNA sequences in eukaryotic chromosomes

-attach long gene rich chromatin loops to non-histone protein structural scaffolds before higher level folding

Front 95

Heterochromatin

Back 95

-regions of chromatin that remain in higher level structure (condensed)

-tend to be rich in repetitive DNA, poor in genes

-usually found in telomeres and centromeres

-not transcriptionally active

-close to nuclear pores

Front 96

Euchromatin

Back 96

-regions of chromatin that completely decondense

-delicate/thread like

-gene rich

-transcriptionally active

Front 97

Centromeres

Back 97

-specific sequence (CEN) in yeast

-78-86bp

-A/T rich

-contain nucleosomes w/ special form of histone H3

-bound by complex of proteins to spindle fibers

Front 98

Telomeres

Back 98

-stabilizing

-prevent end of chromosome from shortening after every DNA replication (telomerase action)

Front 99

Contiguous Sequences (contigs)

Back 99

-multiple consensus fragment sequences

-gaps in between

-can reconstruct a scaffold (what DNA molecule would look like)

Front 100

Next-Gen Sequencing

Back 100

-high throughput

-microfluidics

-fixed synthesis

-high resolution microscopy

-read length is longer

Front 101

Telomerase

Back 101

-enzyme that adds nucleotides to telomeres

-catalyzes reverse transcription to prevent lagging strand from shortening during DNA replication

Front 102

ATP Sulfurylase

Back 102

-converts pyrophosphate into ATP in presence of adenosine 5' phosphosulfate during pyrosequencing

Front 103

Luciferase

Back 103

-catalyzes conversion of luciferin to oxyluciferin during pyrosequencing

Front 104

Apyrase

Back 104

-nucleotide degrading enzyme

-continuously removes dNTPs and excess ATP after each cycle of pyrosequencing

Front 105

BLAST

Back 105

-finds regions of similarity in biological sequences

-uses blastin to extend match even if the inbetween are not complementary

-allows for short gaps in alignment

Front 106

NF1

Back 106

-neurofibromatosis

-mutations lead to multiple tumours in PNS which result in protuberances in skin (elephant man syndrome)

Front 107

Ira

Back 107

-GTPase accelerating protein that regulates Ras (controls cell replication and differentiation)

Front 108

Orthologous genes

Back 108

-slightly different genes with the same function in different species

-ex. alpha-tubulin in different species

Front 109

Paralogous

Back 109

-similar genes with different functions

-ex. alpha and beta tubulin

-result of duplication and divergence

Front 110

C Value

Back 110

-DNA content of organism

-lack of correlation between genome size and biological complexity

Front 111

Open Reading Frame (ORF)

Back 111

-gene coding regions of exons

Front 112

Promoters

Back 112

-control region

-region of DNA where transcription initiation occurs for particular gene

Front 113

Gene Duplication/Conversion

Back 113

-two copies of gene w/ the same function

-one gene will either evolve a new function or degenerate over time (pseudogenes0

Front 114

Minisatellite DNA

Back 114

-simple sequence repeats

-20-50 tandem repeat units (14-100bp in length)

-arrays of 1-5 kbp in length

Front 115

Microsatellites

Back 115

-simple sequence repeats

-repeat units typically 1-4bp in length

-arrays up to 600bp in length (tandem repeat units)

-sometimes found in transcription units

-expansion of microsatellites underlie several neuromuscular diseases

Front 116

Backwards slippage

Back 116

-bulging out of extra repeat

-affects length of single sequence repeats

Front 117

DNA Fingerprinting

Back 117

-hypervariable nature of SSRs can be used in "fingerprinting" protocols

-SSRs amplified by PCR and number of repeats determined by high resolution gel electrophoresis

-paternity determination, criminal identification

-theory: portion of microsatellite DNA is passed down from parents to child

Front 118

Huntington's Disease

Back 118

-mood/cognitive dysfunction

-involuntary movements

-runs in families, becomes more severe in later generations

-longer the CAG repeat sequence becomes due to slippage and extension the more severe it becomes

Front 119

Cloning

Back 119

-take nucleus from differentiated somatic cell and place in germ line cell

-will be reprogrammed

-implant in embryo

-ex. Dolly the sheep

Front 120

DNA libraries

Back 120

-permanent collection of genes obtained and maintained

-multiple fragments in multiple plasmids at once

-can have full representation of genome through individual plasmids

-genomic libraries contain chromosomal DNA

-cDNA libraries are representative of mRNA present in given sample

Front 121

Bacterial Expression Vectors

Back 121

-specialized vectors can be used to overexpress recombinant proteins of interest.

-ex. insulin

Front 122

Lac Operon

Back 122

-in E.coli

-consists of 3 structure genes (A, Y and Z), operator site, promoter and CAP site

-encodes for proteins involved in metabolism of lactose

-presence of glucose and lactose affect transcription of genes

-can be used as a promoter as strategy for regulating gene expression

Front 123

Lac A

Back 123

-part of lac operon

-encodes for beta galactoside transcetylase

Front 124

Lac Z

Back 124

-part of lac operon

-encodes for beta galactosidase which helps with the cleavage of lactose into glucose and galactose

Front 125

Lac Y

Back 125

-part of lac operon

-encodes for beta galactosidase permease which helps with the absorption of lactose through cell membranes

Front 126

Lac Repressor Protein

Back 126

-will bind to operator site downstream of promoter on lac operon if there is no lactose present

-prevents the transcription of the lac operon

Front 127

Allolactase

Back 127

-isomer of lactose

-present when lactose is present

-acts as inducer of transcription of lac operon gene

-binds to lac repressor protein, preventing it from binding to operator site

Front 128

Catabolite activator protein

Back 128

-if no glucose is present in cell and cAMP levels are high it will bind to the CAP site on the lac operon

-increases transcription of gene

Front 129

cAMP

Back 129

-cyclic AMP

-allows Catabolite activator to bind to CAP site on lac operon

-high levels of glucose inhibit the production of cAMP

Front 130

Transient Transfection

Back 130

-transfect eukaryotic cells

-short term, only a few cells

-requires vector w/ promoter, cDNA, viral origins of replication

-transfect cultured cells by lipid treatment or electroporation

-protein expressed from cDNA in plasmid DNA

Front 131

Stable Transfection

Back 131

-long term

-requires antibiotic resistance

-all cells will express gene forever

-requires promoter, cDNA and vector

Front 132

Molecular Probes

Back 132

-allow you to visualize and quantify amount of nucleic acid of interest

-complex mixture of macromolecules

-bind through Watson-Crick b.p

-use single stranded synthetic oglionuclotides w/ complementary sequences

-label (ex. w/ PNK by phosphorylating oglionucleotides)

Front 133

DNA Southern Blotting

Back 133

-technique used to transfer representation of DNA separation through gel electrophoresis onto a solid state matrix

-used to detect specific sequences in DNA sample

-can be used for looking at pedigrees

-looking at banding patterns (polymorphisms) to analyze blood relations

Front 134

RNA Northern Blotting

Back 134

-technique used to transfer representation of RNA separation through gel electrophoresis, onto solid matrix

-allows us to analyze tissue-specific expression and stage-specific expression of certain genes

Front 135

Kd

Back 135

-dissociation constant, strength of protein-protein interaction

=[(Protein A)(Protein B)/(Protein A - Protein B)]

-small constant means proteins have a higher affinity for each other

Front 136

Km

Back 136

-Michaelis Constant

= [S] at Vmax

-efficiency of E to convert S to P

-increase amount of substrate, initially increase Vmax, then plateau

-Vmax = substrate has been incorporated in all enzymes (tells us the affinity of enzyme for substrate)

Front 137

Calcium/Calmodulin

Back 137

-calmodulin: binds Ca2+

-radical conformational change

-recognizes specific regions of proteins to which Ca2+ binds to

-alters protein function in Ca2+ manner

-many proteins require Ca2+ for optimal function

Front 138

GAPs

Back 138

-GTPase activating proteins

-enhance GTPase activity

-shut off GDP-bound state

Front 139

GEFs

Back 139

-displaces GDP by GTP

Front 140

Western Blotting

Back 140

-electrophoresis and transfer of proteins to solid state support

-use of antibodies to bind to proteins

-use of chromogenic detection

-reveals position, size and abundance of protein

Front 141

HA tag

Back 141

-small peptide identified as flu virus

-used in affinity chromatography

-design a DNA molecule w/ sequence that reads through and translates HA peptide

-peptide in protein (tag) will be recognized by antibody

Front 142

RNA Polymerase I

Back 142

-transcription of ribosomal precursor RNA genes in nucleolus

-28S, 5.8S, 18S

Front 143

Eukaryotic Transcription

Back 143

-compartmentalized

-restricted to plastids (plants/algae), mitochondria and nucleus

-RNA Polymerases I, II and III

Front 144

RNA Polymerase II

Back 144

-transcription of all protein coding genes (mRNA)

-some RNA required for splicing (U1-U5)

-other small non coding RNAs

-will start to transcribe at almost any site of DNA

Front 145

RNA Polymerase III

Back 145

-transcription of rRNA outside nucleolus

-transcription of tRNA genes and small stable RNAs (ex. U6-->splicing)

Front 146

CpG regions

Back 146

-mammalian promoter element

-common in constantly expressed genes (do not require rapid activation)

-tends to initiate transcription at multiple sites (sometimes bidirectionally)

Front 147

Proximal Promoter Control elements

Back 147

-close to promoter sequence (-200bp upstream)

-some even in introns

Front 148

Enhancers

Back 148

-distal elements, kilobases upstream from promoter

-sometimes bind to specific proteins to act as upstream activator

-often protein interaction gives rise to major topological change in chromatin

-chromatin loops up bringing enhancer closer to promoter

Front 149

TATA box

Back 149

-present in a lot of promoters

~ - 30bp

-directs transcriptional start

Front 150

TATA-box binding protein (TBP)

Back 150

-binds very strongly to TATA sequence of TATA box

-identified through affinity chromatography

-distorts DNA, bending it by binding to minor groove of proximal region

-even promoters without TATA box use TBP

Front 151

Initiator

Back 151

-TATA box-less promoters

-seems to play role in positioning RNA Pol II for transcription start

Front 152

TFIID

Back 152

-Transcription Factor Class II D

-harbours TBP

-binds TATA box

-conformational changes within promoter

Front 153

TFIIA/TFIIB

Back 153

-two more TFs that interact w/ upstream promoter

Front 154

TFIIF

Back 154

-RNA Pol II is almost always associated w/ TFIIF

Front 155

Core Pre-Initiation Complex

Back 155

-TFIID (w/ TBP), TATA box, TFIIA, TFIIB, RNA Pol II w/ TFIIF (open pre-initiation complex)

-TFIIE joins complex

-TFIIH joins, closing pre-intiation complex

Front 156

RNA Pol I Transcription

Back 156

-core element and upstream element require core factor and upstream activating factor to interact w/ DNA sequences

-requires assembly of factors on Pol I promoter

-Pol I activates downstream transcription

-no ATP required

-TBP required for optimal transcription

Front 157

RNA Pol III Transcription

Back 157

-core elements required for interaction w/ DNA

-no ATP required

-promoters located w/in transcribed regions

-conserved internal promoter elements (A/B boxes)

-promoters recognized by TFIIIB/TFIIIC that recruit Pol III

-TBP also required

Front 158

A/B Boxes

Back 158

-conserved internal promoter elements in Pol III Transcription

-encode structural elements of tRNA

Front 159

C Box

Back 159

-acts as promoter element in 5S gene transcription (recognized by TFIIIA)

-RNA Pol III transcription

Front 160

Activated Transcription

Back 160

-TBP is not enough

-need full complement of all other factors with TBP for activated transcription

-requires information within promoters to recruit general transcription factors in holoenzyme form

-very efficient

-makes basal level of transcription go way above threshold in response to cellular signal

-trans-acting factor also required to interact w/ DNA elements specific to protein

Front 161

Transcriptional Regulation

Back 161

-rate of transcription main factor

Front 162

RNA-seq

Back 162

-gives representation of how many transcripts were present in sample

-does not tell you whether mRNAs are being actively transcribed

Front 163

Global Run On (GRO-seq)

Back 163

-critical for understanding what genes are actively transcribed by RNA Pol II

-uses bromouridine markers
-once RNA Pol II is engaged in elongation it will be progressive before pausing (characteristic of certain regions)

Front 164

ChIP-seq

Back 164

-gives levels of transcription of all transcripts in sample (which ones are more highly transcribed vs those that are not, etc)

-does not account for pausing or RNA Pol II during elongation

-does not differentiate active transcripts

Front 165

TFs as Regulators of Transcription Activation

Back 165

-recognize specific DNA motifs (alpha-helical domain: Recognition Helix)

-interaction w/ major groove of DNA (unlike proteins)

-does not cause conformational change

-recruit general transcription factors

Front 166

Reporter Genes

Back 166

-downstream

-facilitate relative quantification of transcriptional efficacy

-ex. lac operon, GFP, thymidine kinase

-place reporter gene inside control region that is unmanipulated and transfect into cell

-see how well TF may activate reporter gene via linker scanner analysis

Front 167

Linker Scanner Analysis

Back 167

-pinpoint sequence w/ regulatory function

Front 168

Transcription Factors

Back 168

-interact w/ DNA to affect transcription complex (activate/repress)

-modular

-several domains

-general TFs required for basal transcription

-activated transcription uses basal TFs and makes them more efficient (uses activators)

-Recognition Helix reacts w/ major groove

Front 169

Homeodomain Proteins

Back 169

-DNA binding TF

-presence of several TFs that give rise to homeotic transformations (ex. legs replacing antennae)

-human HOX genes are homeobox proteins (similar to antennapedia)

Front 170

Zinc Finger Binding TF

Back 170

-different types exist

-made up of cysteines and histamines

-C2H2 contains 3+ finger units and binds to DNA as a monomer

-C4 usually only has 2 finger units and binds to DNA as a homo/heterodimer

-C6 is variation in which 6 cysteine metal ligands bind to two Zn 2+ ions

-alter transcription in trans acting manner

Front 171

Leucine zipper proteins

Back 171

-stretch of hydrophobic amino acids that form interfaces

-interfaces allow two leucine zippers to join and form dimers

-gives abilitly to interact w/ major groove sites

-can form either hetero or homodimers

-extended alpha-helices

Front 172

Helix-loop-helix proteins

Back 172

-similar to leucine zipper

-have two short alpha helices connected by short loop

-contains hydrophobic amino acids spaced at intervals

Front 173

DNAse Footprinting

Back 173

-proteins interact w/ DNA to protect from degradation by DNAse

-use to determine the protected sequence of DNA

Front 174

Cooperative Binding of TFs

Back 174

-TFs of unrelated classes can also bind cooperatively

-ex. AP1 can bind to DNA but doesn't activate transcription that well until NFAT also binds to same site on DNA

-increases diversity in gene regulation

Front 175

Carboxy-Terminal Domain (CTD)

Back 175

-only on RNA Pol II

-critical for capping, splicing, polyadenylation, export

-52 repeats of heptopeptide

-phosphorylated on Ser-5 by protein kinase to transition initiation to elongation

-second phosphorylation on Ser-2 switches RNA Pol II to full on elongation

Front 176

5' Cap

Back 176

-7'methylguanylate

-added to 5' terminal nucleotide through unusual 5'-5' linkage

-protects pre-mRNA, facilitates nuclear export, recognition by TFs

-required for efficient translation initiation

Front 177

Introns

Back 177

-not junk (can encode regulatory information)

-discovered due to discrepancy between mRNA and gene size

-intron sequences in DNA loop out

-intron borders are highly conserved (GU and 5' splice site and AG at 3' splice site)

-toward 3' end of introns there are critical sequences for downstream events

-pyrimidine rich region

-conserved branch point A

Front 178

Spliceosome

Back 178

-small nuclear RNAs essential for spliceosome functionality (5 snRNPs)

-U1: interacts w/ 5' intron border (shares limited sequence homology)

-U2: defines branch point A

-where U2 does not pair w/ branch point A bulges out

Front 179

Self Splicing Introns

Back 179

-RNAs capable of catalyzing trans-esterification reactions (ribozymes)

-does not need spliceosome

-Group I self splicing introns: nuclear rRNA genes of protozoans

-Group II self splicing introns: some rRNA/tRNA genes in mitochondria and chloroplasts

-exception not rule

Front 180

RNA Binding proteins

Back 180

-bind through specific domains to form ribonucleoprotein complex (RNP)

-RNAs are rarely naked

Front 181

Debranching enzyme

Back 181

-linearizes lariat structure after intron splicing to allow enzymes to degrade intron RNA

Front 182

SR proteins

Back 182

-RNA binding proteins w/ RRM domains and protein-protein interaction domains

-bind exonic splicing enhancer sequences to exons

-help splicing machinery understand where intron-exon boundaries should be

-facilitates binding of U1 to 5' splice site and U2 to branch point A

-form cross-exon recognition complex w/ proteins and snRNAs

Front 183

U2AF

Back 183

-splicing factor

-helps w/ splicing efficiency

-two subunits

-small subunit binds to AG at 3' intron-exon boundary

-helps U2 snRNA/snRNP sit down on correct branch point A

-larger subunit interacts w/ polypyrimidine tract

-marks where 3' extremity is

Front 184

Phosphorylation of CTD

Back 184

-unique to Class II transcription

-Ser5 phosphorylation mediates capping by capping enzyme

-Ser2 is critical for fast elongation

-factors required for splicing, polyadenylation and export bind to phosphorylated CTD

-this places them in close proximity to pre-mRNA emerging from advancing transcription bubble

-can just jump off CTD at any time and interact w/ pre-mRNA

Front 185

Exonic Splicing Enhancers

Back 185

-sequences within exon that promote exon joining after splicing

-decorated by SR proteins

Front 186

Alternative Splicing

Back 186

-can transcribe one gene yet give rise to several different proteins depending on how/what you splice

-ex. fibronectin pre-mRNA

-fibroblasts: 2 exons play role in adhesion

-fibronection: no sticky function b/c it is secreted in blood stream so those exons are not included

Front 187

Sxl

Back 187

-sex determination in Drosophila

-under transcriptional control

-expressed in early female embryos only

-male embryos do not produce sex lethal proteins

Front 188

Polyadenylation

Back 188

-co-transcriptional

-required for stability

-all mRNA transcripts are polyadenylated (except histone mRNA)

Front 189

Poly A Polymerase (PAP)

Back 189

-catalyzes formation of Poly A tail

-activates cleavage reaction

-adds ~12 A residues to 3' end

Front 190

Poly A Binding Protein II (PABPII)

Back 190

-recognizes polyadenylation complex after first slow phase and catalyzes the rapid addition of ~200 A residues to the 3' end

Front 191

RNA Editing

Back 191

-sequence of mature mRNA differs from sequence of coding region of genomic DNA

-widespread in mitochondria and plasmids

-pre-mRNA is affected by changes in single nucleotides within sequences of pre-mRNAs

-ex. deamination turns C residue to U (can encode stop codon!)

Front 192

tRNA

Back 192

-must be able to read codon

-must be able to covalently link to amino acids

-clover leaf-like structure due to b.p between some regions (stems) and non bp regions (loops)
-must be processed post-transcriptionally

Front 193

rRNA

Back 193

-accounts for 80% of total cellular RNA

-pre-rRNA transcription units are arranged in reptitive clusters

-fold into highly conserved stem-loop structures

Front 194

Bacterial Ribosome

Back 194

-23S + 5S + 31 proteins = 50S large subunit

-16S + 21 proteins = 30S small subunit

together = 70S complex

Front 195

Eukaryote Ribosome

Back 195

-28S hybridized w/ 5.8S + 5S + 50 proteins = 60S large subunit

-18S + 33 proteins = 40S small subunit

together = 80S complex

-roughly the same size as bacterial

-3 sites (A, P, E)

Front 196

eIF5B

Back 196

-recruits 50S large subunit during initiation of eukaryotic translation

Front 197

tRNA initiator

Back 197

-bound to Met

-binds to P site in 43S pre-initiation complex

-different from Met-tRNA for elongation (binds to A site)

Front 198

Elongation Factor 1 alpha (EF1a)

Back 198

-bound to GTP

-interacts w/ A site during elongation of eukaryotic translation

-if anticodon and codon at A site match GTP will be hydrolyzed

-EF1a-GDP leaves

Front 199

Elongation Factor 2 (EF2)

Back 199

-bound to GTP

-hydrolyzes GTP to translocate tRNAs one site forward during eukaryotic translation

Front 200

Eukaryotic Release Factor 1 (eRF1)

Back 200

-mimics aminoacyl-tRNAs

-binds to A site

Front 201

Eukaryotic Release Factor (eRF3)

Back 201

-bound to GTP

-hydrolyzes GTP when eRF1 binds to A site

-allows for release of polypeptide chain at P site and tRNA at E site

Front 202

mRNA Stability

Back 202

-more stable mRNAs are more actively transcribed and more efficiently translated

-longer Poly A tails provide stability because it takes longer for them to be digested by exonucleases (coding info is safe)

-PABP blocks exosome from chewing mRNA 3' to 5'

-longer tails means greater interaction between PABP and eIF4G to form loops that favour re-initiation of translation

Front 203

Protein Folding

Back 203

-polypeptides may have to be folded as they are being synthesized

-have to be protected

-ex. large hydrophobic domains can be exposed during translation (crunching up into ball)

-proteins fold into lowest energy state conformation

-folding is critical for proper function

-many denatured proteins can refold

Front 204

Heat Shock Proteins

Back 204

-molecular chaperones

-ensure polypeptides fold properly

-proteins can be used immediately

-ex. Hsp70 and Hsp90

-use ATP to bind to specific subunits

-ATP hydrolysis releases folded polypeptide

-if protein is not properly folded it undergoes more cycles

-Hsp levels increase in response to heat shock/cellular stress

Front 205

Chaperonins

Back 205

-giant macromolecular machines entirely devoted to folding proteins

-made up of identical subunits (2 stacked tires)

-ex. GroEL in bacteria and TriC in humans

-take client protein into upper chamber

-use ATP to enhance folding within upper chamber (tight conformation)

-ATP hydrolysis causes conformational change (tight to relaxed)

-correctly folded protein released

-will go through more cycles if not folded properly

Front 206

Pathologies Associated w/ Inappropriate Folding of Protiens

Back 206

-associated w/ improperly folded proteins forming aggregates that form "plaques"

-ex. Alzheimers, Huntingons, Kuru

-cause or death?

Front 207

Prion proteins

Back 207

-proteniaceous infectious agent

-expressed in everyone

-conformer (alpha helices) and non conformer (beta sheets)

-non conformer is infectious, interacts w/ conformer and changes its structure to adopt beta sheet structure

-formation of plaques that grow into fibrils

-holes in brain

-cellular dysfunction

-ex. Kuru

Front 208

Kuru

Back 208

-neurodegenerative disease, Prions

-"Laughing Death"

-loss of limb control, crossing of eyes

-Fore people

-transmitted through ritualistic cannibalism

Front 209

ER Signal Sequence

Back 209

-present in first few amino acid sequence of mRNA

-sends mRNA to rough ER

-ribosomes embedded into rough ER translate polypeptide directly into ER

-proteins that require disulfide bridges must be synthesized in ER

Front 210

ER Chaperones

Back 210

-ex. BiP

-make sure proteins transiting through ER and properly folded

Front 211

Nuclear Pore Complex (NPC)

Back 211

-"basket"

-highly ordered structure

-cytoblasmic filaments

-nuclear basket

-about 125 mega Daltons (30x larger than ribosome)

-small molecules diffuse freely through

-larger molecules and multlicellular complexes require active transport

Front 212

FG Nucleoporins

Back 212

-critical for movement of complexes across pore

-FG repeat domains

-confer hydrophobicity to protein

-facilitate interactions between cargo and transporter going through nuclear pore

Front 213

Nuclear Localization Signal (NLS)

Back 213

-short a.a sequence

-protein is recognized by/interacts w/ transporter

-essential for nuclear localization

Front 214

Ran

Back 214

-monomeric G protein that exists in two forms

-bound to GTP (active)

-bound to GDP (inactive)

-GAPs act as off switch (hydrolyze GTP)

-GEFs act as on switch (displace GDP w/ GTP)

-required for transport of tRNA, rRNA, and most proteins

-mRNA is transported Ran-independantly

Front 215

Nuclear Transport Receptors

Back 215

-importins

-bind to NLS domains present on cargo proteins

-facilitate transport through pore by association w/ FG nucleoporins

Front 216

Nuclear Export Sequence (NES)

Back 216

-recognized by Exportin 1

-amino acid sequence

Front 217

Exportin 1

Back 217

-for Ran-dependent nuclear export

-recognizes NES

Front 218

Exportin t

Back 218

-exports tRNAs out of nucleus

Front 219

mRNA Exporter

Back 219

-consists of two subunits (Nxf1 and Nxt1)

-bind to RNA cooperatively w/ specific mRNP proteins (including SR)

-form domain that interacts w/ FG nucleoporins

-acts as both importin/exportin

-Ran-independent

-5' end of mRNA must enter NPC first

-only fully mature mRNAs get exported

-unprocessed mRNA will be degraded upon entering cytoplasm

Front 220

Mechanisms for Translation Inhibition

Back 220

-phosphorylation of eIF2 alpha (translational complex not formed)

-proteins bind to IFs (proper formation of eIF4 blocked, cannot form loop configuration for translation/reinitiation)

-removal of Poly A tail by exonuclease activity (destabilizes mRNA)

Front 221

Iron Response Element Binding Protein (IRE-BP)

Back 221

-critical for maintaining homeostatic intracellular levels of iron

-post transcriptional regulation

-in high iron conditions it is inactive and cannot interact w/ RNA

-in low iron conditions it is active and itneracts w/ stem-loop IRE structures on mRNA

Front 222

Mammalian Transfer Receptor (TfR)

Back 222

-needed for import into cell

-stability of receptor is regulated to intracellular iron concentration

-has IREs on 3' UTR of mRNA

Front 223

Ferritin

Back 223

-intracellular protein that binds to iron ions

-prevents accumulation of toxic levels of free iron ions

Front 224

mRNA Translation Inhibition in Drosophila Embryo Development

Back 224

-early events in embryogenesis specifies axis/poles of egg

-hunchback protein: specifies anterior

-nanos: specifies postieor

-hunchback mRNA present throughout egg

-nanos inhibits the translation of hunchback protein in posterior region

-nanos is RNA binding protein that binds to hunchback mRNA

Front 225

Post-Translational Regulation

Back 225

-protein modifications:

-phosphorylation

-glycosylation

-addition of lipids

-methylation/acetylation

-protein stability:

-proteolysis (ubiquitin)

Front 226

Phosphorylation

Back 226

-protein kinases accept two substrates

-transfer phosphate from one to another

-can be responsible for radical conformational changes (active/inactive)

-addition of phosphate adds negative charge (can cause gel shift)

-can expose domains (nuclear localisation)

Front 227

Proteolysis

Back 227

-degradation of proteins

-sometimes regulated manner

-ex. beta catenin and cylcins

-misregulation of beta-catenin can cause accumulation of cells

-misregulation of cyclins can cause constant cell divisions

-way proteins cycle throughout cell life

Front 228

Ubiquitin

Back 228

-small 76 amino acids long polypeptide

-can be covalently linked to lysine residues

-foundding member of family of polypeptide modifiers

-add one ubiquitin to lysine residue and then more ubiquitins to form chains

-add mass to protein

-polyubiquilaytion can target proteins for degradation via proteasome

Front 229

MAT locus in yeast

Back 229

-regulates mating type switching in yeast

-only mating type info transferred into MAT locus will be expressed

-mating type info in loci on ends (HML/HMR) are silenced (genes cannot be expressed)

-genetic analysis indicates that mutations in histones (tails) alleviates silencing of genes in HML/HMR

Front 230

Telomere Silencing Effect

Back 230

-genes placed in proximity to telomeres will be silenced

-mutations in histones can alleviate silencing effect

Front 231

RAP1

Back 231

-factor required for repression of silent mating type loci

-bind to DNA in region of silencer

-recruits other proteins to act in major complex to change acetylation status of histones in silenced areas

-also in telomeres

Front 232

SIR1

Back 232

-interacts w/ RAP1 in both telomeres and silent mating type loci

-sets up scaffold to be recognized by more SIRs (2,3,4)

Front 233

SIR2

Back 233

-protein that recognizes RAP1/SIR1 bound to telomeric/silent mating type loci

-sets up major complex w/ SIR 3 and 4

-has enzyme activity that removes acetyl groups from histones (histone acetylase)

Front 234

Histone Deacetylation

Back 234

-removal of acetyl groups from histone tails

-histone interactions w/ DNA backbone are altered

-usually deacetylation (increase positive charge) will cause strong interaction w/ DNA and very tight structure

-repression of transcription/translation/gene expression

Front 235

Histone Acetylation

Back 235

-addition of acetyl group to histone tails

-neutralizes histone positive charge

-weakens bonds between histone and DNA

-loosens chromatin

-allows basal TFs to interact w/ DNA more actively

-activator TFs usually recruit histone acetyl transferases (HATs)

Front 236

Rpd3

Back 236

-in yeast

-subunit recruited by DNA binding TF (repressor)

-has deacetylase function

-shuts down transcription

-orthologs in higher eukaryotes

-also exists in co-repressor complexes (many containing Sin3p and Ume6p)

Front 237

Sin3p

Back 237

-in corepressor complexes

-associated w/ histone deacetylation

Front 238

Ume6p

Back 238

-involved in corepressor complexes

-required for specific targeting (binds URS)

Front 239

Co-activators

Back 239

-combine w/ DNA binding TFs that bring them down to loci to change

-hyperacetylation function

Front 240

Methylation of H3 K4

Back 240

-associated w/ positive effect on transcription

-H3 K4 mono-methylation accumulates around enhancers

-H3 K4 polymethylation associated w/ actively transcribed genes

Front 241

Methylation of H3 K9

Back 241

-associated w/ shut down of transcription

-seen in heterochromatin

Front 242

Dosage Compensation

Back 242

-ex. only one X chromosome is needed for survival

-females inactivate one X during embryogenesis

-random decision as to which X chromosome is inactivated

-always the same X chromosome inactivated after cell division

-cell will always inactivate extra X chromosomes

Front 243

Calico cats

Back 243

-coat colour on X chromosome

-inactivation of X chromosome is altered providing coat colour information from two different X chromosomes (black and brown)

-therefore: all calico cats are female

Front 244

XIST

Back 244

-locus that encodes for long non-coding RNA produced after X inactivation

-gives rise to heterochromatin spreading and decrease in gene expression

-required for establishment of inactivation but not maintenance

Front 245

Epigenetic Traits

Back 245

-transmitted to subsequent generations independently of DNA sequence itself

-changes in chromatin propagated

-ex. Inactive X, development restrictions, imprints (DNA methylation)

Front 246

Centromeric Region Silencing

Back 246

-in yeast small RNAs are required for silencing mechanism (dsRNAs)

-dsRNA nucleates complex that involves several proteins involved in the generation of H3 K9 (repressive)

Front 247

RNA-mediated Interference

Back 247

-introduction of RNA antisense molecule into organism giving rise to complete/near complete elimination of corresponding sense RNA translation

-does not affect transcription of gene

-destroys mRNAs

-highly conserved

Front 248

Transgenic dsRNAs

Back 248

-dsRNA introduced as transgene causes loss of function phenotypes

Front 249

t7 RNA Polymerase Promoters

Back 249

-make linear PCR template by driving transcription in opposite directions on each side of target sequence

Front 250

siRNA

Back 250

-small interfereing RNA

-dsRNA cleaved by dicer

-interacts w/ Argonaute proteins in RISC

Front 251

miRNAs

Back 251

-small RNAs that act in antisense manner

-everywhere/everything

-metabolism, tissue growth, neural development, developmental timing, etc is all mediated by miRNAs

Front 252

ceRNA

Back 252

-competitive endogenous RNA

-can be products of pseudogenes, long non-coding RNAs, circular RNAs

-target sites for miRNAs

-can bind to miRNAs and titrate away from mRNA target

Front 253

miRNA sponges (circRNA)

Back 253

-small circular RNA molecules w/ miRNA binding sites

Front 254

Peewee RNAs

Back 254

-associated w/ Argonaute proteins responsible for silencing/licensing of chromatins

Front 255

CRISPR

Back 255

-region of bacterial genome that incorporates chunks of viral DNA after viral infection

-transcribes crRNA during next bacteriophage attack

Front 256

sgRNA

Back 256

-tracerRNA + crRNA

-corresponds to any targeting locus/sequence of interest that is fused to tracrRNA

-recruits Cas to region of viral genome

Front 257

Cas

Back 257

-bacterial endonuclease that makes double strand breaks in viral genome during bacterial acquired immune response

-best characterized is Cas9