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257 Cards in this Set

  • Front
  • Back


-just the nitrogenous base no phosphate groups

Amino Acid Isomers

-D and L

-humans have adapted to use L isomers


-can form disulfide bonds with another cysteine between sulfhydryl groups

-stabilizes folding structure


-small and compact due to symmetrical hydrogens


-rigid ring structure

Coiled-coil motif

-derived from fibrous proteinso

Motifs and Domains

motifs: combinations of secondary structures

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

EF hand/helix-loop-helix motif

-ionic bonds involving proteins and Ca2+

Zinc-finger Motif

-series of histamines/cysteines

B form of DNA

-most commonly found in cells

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


-occurs in low humidity/dehydrated samples of B DNA

-shorter and more compact

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


-short DNA molecules that adopt left handed helix configuration

-transiently formed after transcription

-has been found in cells


-two alpha and two beta subunits plus a sigma factor

Sigma Factor

-essential role in selecting site of transcription initiation

-finds promoter

Aminoacyl-tRNA synthetase

-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)

Initiation Factor 1

-associates with 30S subunit

-assists with loading preinitiation complex onto mRNA

-later recruits the 50S ribosomal subunit

Initiation Factor 3

-associates with 30S subunit

-assists loading of preinitiation complex onto mRNA

Initiation Factor 2-GTP

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

23S rRNA


-carries out peptidyltransferase reaction during elongation of peptide chain

Shine-Delgarno box

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

-just before AUG sequence

-required for bacterial translation initiation

Release Factors 1 and 2

-mimic tRNAs


-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


-eukaryotic initiation factor

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

Dimeric Guanylyltransferase

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


-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

KOZAK sequence


-in mammalian translation

-relatively conserved (mainly A and G)


-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

Elongation Factor 2

-bound to GTP

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


-termination release factors in eukaryotes

-mimics aminoacyl-tRNAs


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

-associated with eRF1 bound to A site


-dissociates post-termination complex


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

Large T-antigen

-encoded by SV40 virus


-opens DNA at high rates (helicase)

Replication Protein A

-bind to single stranded DNA

-keeps template in optimal configuration for DNA Pol A

Origin of Replication Complex

-six subunit protein

-binds to replication origins

-associates w/ proteins to load helicases


-proliferating cell nuclear antigen

-homotrimetric protein

-binds around polymerase

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


-relieves supercoils in DNA


-synthesizes short RNA sequences called primers

DNA Polymerase Alpha

-recognizes primer area well

-not very good at extending

-initially required for leading and lagging synthesis

DNA Polymerase Epsilon

-required for full extension of DNA leading strand

-proofreads errors made in base pairing with Polymerase Delta

DNA Polymerase Delta

-fully extends DNA lagging strand

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

-participates in proofreading with Pol E

DNA Polymerase Beta

-participates in base excision repair

-fills gaps left by removed incorrect base

Ribonuclease H/FEN-1

-displace RNA component of 5' ends of Okazaki fragments

DNA glycosylase

-participates in base excision repair

-hydrolyzes bond between mispaired base and sugar phosphate backbone


-cuts sugar phosphate backbone in base excision repair


AP lyase


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



-cuts sugar backbone during mismatch excision repair


-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)

MLH1 Endonuclease

-participates in mismatch excision repair

Protein 23B

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

-allows for opening of helix


-protein complex

-scans DNA for distortions

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


-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)


-acts as endonuclease in nucleotide excision repair

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


-DNA dependent protein kinase

-recognizes breaks in DNA strand during end-joining



-binds to ends of break in DNA strand during endjoining


-recognizes 3' overhangs during homologous recombination

-facilitates strand invasion

-in prokaryotes


-recognizes 3' overhangs during homologous recbomination

-facilitates strand invasion

-in eukaryotes


-most common vector used in recombinant DNA technology

-circular double stranded DNA found in bacteria/lower eukaryotes



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


-terminal nucleotides, dideoxynucleotides

-no hydroxyl group on 3' carbon of sugar

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


-mobile gene elements found in prokaryotes and eukaryotes


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

DNA Transposons

-"cut and paste" mechanism

-autonomous (have transposase gene) and non autonomous

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


-act like retroviruses (act through RNA intermediate)

-encode for reverse transcriptase

LTR Retrotransposons


-target site direct repeats (5-10bp)

-Long Terminal Repeats (250-600bp)

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



-encoded by retroviral genome

-group specific antigen


-encoded by retroviral genome

-reverse transcriptase function


-encoded by retroviral genome

-envelope protein

-allows retrovirus to leave host cell


-Long Interspersed Elements

-no LTRs

-6-8kb in length

-present in L1

-ORF1 and ORF2



-Open Reading Frame 1

-on LINEs

-codes for RNA-binding protein


-Open Reading Frame 2

-on LINEs

-codes for reverse transcriptase and DNA endonuclease


-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

Processed Pseudogenes

-mobilized protein-coding genes

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

-lack introns and control regions

-often not expressed


-"domesticated transposon" gene

-derived from transposase gene

-encodes for recombinases important for assembly of human immunoglobin genes


-"domesticated transposon" gene

-encodes for DNA binding proteins that mediate placental cell fusion

-derived from LTR-retrotransposons

Insertion Mutagenesis

-transposon lands in gene

-causes gene to become non-functional

-ex. maize , snapdragon, wine grape colours

Gene and Segmental Duplication

-gene duplication results from uneven cross over

-ex. alpha/beta hemoglobin genes

Exon Shuffling

-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

Cis-regulatory Module

-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


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


-host genomic region within a DNA transposon

Organellar DNAs

-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)

Mitochondria DNA

-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

DNA Barcoding

-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


-nucleo-protein complex

-when cells are not dividing DNA is found in chromatin

-extended and compact forms

"Beads on a String"

-extended form of chromatin

-bead = nucleosome

-string = 10-90bp of linker DNA

Solenoid Model of 30nm fiber

-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

Two start Helix model of 30nm fibre

-nucleosomes form stack of "coins"

-stacks form helices


-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


-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)


-histone type

-highly conserved among distantly related species

-has C-terminal tails


-histone type

-highly conserved

-N-terminal tails


-histone type


Histone Code

-particular combinations of modifications collectively found in chromatin

-can create/remove chromatin-protein binding sites

-determine which regions of genes in chromatin are transcribed


-specific DNA sequences in eukaryotic chromosomes

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


-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


-regions of chromatin that completely decondense

-delicate/thread like

-gene rich

-transcriptionally active


-specific sequence (CEN) in yeast


-A/T rich

-contain nucleosomes w/ special form of histone H3

-bound by complex of proteins to spindle fibers



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

Contiguous Sequences (contigs)

-multiple consensus fragment sequences

-gaps in between

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

Next-Gen Sequencing

-high throughput


-fixed synthesis

-high resolution microscopy

-read length is longer


-enzyme that adds nucleotides to telomeres

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

ATP Sulfurylase

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


-catalyzes conversion of luciferin to oxyluciferin during pyrosequencing


-nucleotide degrading enzyme

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


-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



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


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

Orthologous genes

-slightly different genes with the same function in different species

-ex. alpha-tubulin in different species


-similar genes with different functions

-ex. alpha and beta tubulin

-result of duplication and divergence

C Value

-DNA content of organism

-lack of correlation between genome size and biological complexity

Open Reading Frame (ORF)

-gene coding regions of exons


-control region

-region of DNA where transcription initiation occurs for particular gene

Gene Duplication/Conversion

-two copies of gene w/ the same function

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

Minisatellite DNA

-simple sequence repeats

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

-arrays of 1-5 kbp in length


-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

Backwards slippage

-bulging out of extra repeat

-affects length of single sequence repeats

DNA Fingerprinting

-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

Huntington's Disease

-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


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

-will be reprogrammed

-implant in embryo

-ex. Dolly the sheep

DNA libraries

-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

Bacterial Expression Vectors

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

-ex. insulin

Lac Operon

-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

Lac A

-part of lac operon

-encodes for beta galactoside transcetylase

Lac Z

-part of lac operon

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

Lac Y

-part of lac operon

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

Lac Repressor Protein

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

-prevents the transcription of the lac operon


-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

Catabolite activator protein

-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


-cyclic AMP

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

-high levels of glucose inhibit the production of cAMP

Transient Transfection

-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

Stable Transfection

-long term

-requires antibiotic resistance

-all cells will express gene forever

-requires promoter, cDNA and vector

Molecular Probes

-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)

DNA Southern Blotting

-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

RNA Northern Blotting

-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


-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


-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)


-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


-GTPase activating proteins

-enhance GTPase activity

-shut off GDP-bound state


-displaces GDP by GTP

Western Blotting

-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

HA tag

-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

RNA Polymerase I

-transcription of ribosomal precursor RNA genes in nucleolus

-28S, 5.8S, 18S

Eukaryotic Transcription


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

-RNA Polymerases I, II and III

RNA Polymerase II

-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

RNA Polymerase III

-transcription of rRNA outside nucleolus

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

CpG regions

-mammalian promoter element

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

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

Proximal Promoter Control elements

-close to promoter sequence (-200bp upstream)

-some even in introns


-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

TATA box

-present in a lot of promoters

~ - 30bp

-directs transcriptional start

TATA-box binding protein (TBP)

-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


-TATA box-less promoters

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


-Transcription Factor Class II D

-harbours TBP

-binds TATA box

-conformational changes within promoter


-two more TFs that interact w/ upstream promoter


-RNA Pol II is almost always associated w/ TFIIF

Core Pre-Initiation Complex

-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

RNA Pol I Transcription

-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

RNA Pol III Transcription

-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

A/B Boxes

-conserved internal promoter elements in Pol III Transcription

-encode structural elements of tRNA

C Box

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

-RNA Pol III transcription

Activated Transcription

-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

Transcriptional Regulation

-rate of transcription main factor


-gives representation of how many transcripts were present in sample

-does not tell you whether mRNAs are being actively transcribed

Global Run On (GRO-seq)

-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)


-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

TFs as Regulators of Transcription Activation

-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

Reporter Genes


-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

Linker Scanner Analysis

-pinpoint sequence w/ regulatory function

Transcription Factors

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


-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

Homeodomain Proteins

-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)

Zinc Finger Binding TF

-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

Leucine zipper proteins

-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

Helix-loop-helix proteins

-similar to leucine zipper

-have two short alpha helices connected by short loop

-contains hydrophobic amino acids spaced at intervals

DNAse Footprinting

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

-use to determine the protected sequence of DNA

Cooperative Binding of TFs

-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

Carboxy-Terminal Domain (CTD)

-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

5' Cap


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

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

-required for efficient translation initiation


-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


-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

Self Splicing Introns

-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

RNA Binding proteins

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

-RNAs are rarely naked

Debranching enzyme

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

SR proteins

-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


-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

Phosphorylation of CTD

-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

Exonic Splicing Enhancers

-sequences within exon that promote exon joining after splicing

-decorated by SR proteins

Alternative Splicing

-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


-sex determination in Drosophila

-under transcriptional control

-expressed in early female embryos only

-male embryos do not produce sex lethal proteins



-required for stability

-all mRNA transcripts are polyadenylated (except histone mRNA)

Poly A Polymerase (PAP)

-catalyzes formation of Poly A tail

-activates cleavage reaction

-adds ~12 A residues to 3' end

Poly A Binding Protein II (PABPII)

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

RNA Editing

-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!)


-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


-accounts for 80% of total cellular RNA

-pre-rRNA transcription units are arranged in reptitive clusters

-fold into highly conserved stem-loop structures

Bacterial Ribosome

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

-16S + 21 proteins = 30S small subunit

together = 70S complex

Eukaryote Ribosome

-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)


-recruits 50S large subunit during initiation of eukaryotic translation

tRNA initiator

-bound to Met

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

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

Elongation Factor 1 alpha (EF1a)

-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

Elongation Factor 2 (EF2)

-bound to GTP

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

Eukaryotic Release Factor 1 (eRF1)

-mimics aminoacyl-tRNAs

-binds to A site

Eukaryotic Release Factor (eRF3)

-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

mRNA Stability

-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

Protein Folding

-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

Heat Shock Proteins

-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


-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

Pathologies Associated w/ Inappropriate Folding of Protiens

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

-ex. Alzheimers, Huntingons, Kuru

-cause or death?

Prion proteins

-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


-neurodegenerative disease, Prions

-"Laughing Death"

-loss of limb control, crossing of eyes

-Fore people

-transmitted through ritualistic cannibalism

ER Signal Sequence

-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

ER Chaperones

-ex. BiP

-make sure proteins transiting through ER and properly folded

Nuclear Pore Complex (NPC)


-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

FG Nucleoporins

-critical for movement of complexes across pore

-FG repeat domains

-confer hydrophobicity to protein

-facilitate interactions between cargo and transporter going through nuclear pore

Nuclear Localization Signal (NLS)

-short a.a sequence

-protein is recognized by/interacts w/ transporter

-essential for nuclear localization


-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

Nuclear Transport Receptors


-bind to NLS domains present on cargo proteins

-facilitate transport through pore by association w/ FG nucleoporins

Nuclear Export Sequence (NES)

-recognized by Exportin 1

-amino acid sequence

Exportin 1

-for Ran-dependent nuclear export

-recognizes NES

Exportin t

-exports tRNAs out of nucleus

mRNA Exporter

-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


-5' end of mRNA must enter NPC first

-only fully mature mRNAs get exported

-unprocessed mRNA will be degraded upon entering cytoplasm

Mechanisms for Translation Inhibition

-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)

Iron Response Element Binding Protein (IRE-BP)

-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

Mammalian Transfer Receptor (TfR)

-needed for import into cell

-stability of receptor is regulated to intracellular iron concentration

-has IREs on 3' UTR of mRNA


-intracellular protein that binds to iron ions

-prevents accumulation of toxic levels of free iron ions

mRNA Translation Inhibition in Drosophila Embryo Development

-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

Post-Translational Regulation

-protein modifications:



-addition of lipids


-protein stability:

-proteolysis (ubiquitin)


-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)


-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


-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

MAT locus in yeast

-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

Telomere Silencing Effect

-genes placed in proximity to telomeres will be silenced

-mutations in histones can alleviate silencing effect


-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


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

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


-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)

Histone Deacetylation

-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

Histone Acetylation

-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)


-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)


-in corepressor complexes

-associated w/ histone deacetylation


-involved in corepressor complexes

-required for specific targeting (binds URS)


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

-hyperacetylation function

Methylation of H3 K4

-associated w/ positive effect on transcription

-H3 K4 mono-methylation accumulates around enhancers

-H3 K4 polymethylation associated w/ actively transcribed genes

Methylation of H3 K9

-associated w/ shut down of transcription

-seen in heterochromatin

Dosage Compensation

-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

Calico cats

-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


-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

Epigenetic Traits

-transmitted to subsequent generations independently of DNA sequence itself

-changes in chromatin propagated

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

Centromeric Region Silencing

-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)

RNA-mediated Interference

-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

Transgenic dsRNAs

-dsRNA introduced as transgene causes loss of function phenotypes

t7 RNA Polymerase Promoters

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


-small interfereing RNA

-dsRNA cleaved by dicer

-interacts w/ Argonaute proteins in RISC


-small RNAs that act in antisense manner


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


-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

miRNA sponges (circRNA)

-small circular RNA molecules w/ miRNA binding sites

Peewee RNAs

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


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

-transcribes crRNA during next bacteriophage attack


-tracerRNA + crRNA

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

-recruits Cas to region of viral genome


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

-best characterized is Cas9