Biology 2960 Exam 3
Adjacent genes encode Lacl= lac repressor
- lac repressor= always in low levels - binds to lacO site on DNA, inhibits transcription - binds to lactose (inducer) - when bound to lactose repressor does not bind lacO
Nanopore sequencing
- no DNA synthesis, instead direct detection - ex: minion device: enzyme unwinds DNA feeding one strand through a protein pore. the unique shape of each DNA base causes a characteristic disruption in electrical current, providing a readout of the underlying sequence
transcription of a mammalian Pol 2 gene
- only 1/5 % codes for proteins
Lactose operon
- operon: 2+ adjacent structural genes that are coordinately controlled by transcription from a single promoter - a single mRNA that harbors multiple protein coding seq= polycistronic RNA - structural genes encode B galactosidase, permease, transacetylase - regulatory DNA at 5'end: promoter (p) and operator (o)
A typical prokaryotic operon
- operons= multiple ORF present in one transcription- each has on ATG and stop -transcription starts at specific site= "+" * determined by promoter - transcription termination specified - no introns: 1 trx= mRNA
"F" plasmids and partial diploids
- partial diploids= normal chromosome and an F' plasmid with 2nd copy of small DNA segment - ex: add 2nd copy of lacl gene - allows for scientists to examine dominant/recessive relationships between alleles and to ask if relative position of alleles is important
Catabolite "Repression" via Cap
- pos regulation of catabolic operons - E. coli prefers use of glucose over lactose - extra layer of regulation ensures that bacteria does not invest energy in utilizing other sugars until glucose is exhausted - glucose is a catabolite of lactose
Histone Lysine Acetylation
- weakens DNA binding - DNA is acidic because of neg charged phosphates - histones are basic because of abundance (+) lysine and arginine aa residues - lysine in amino terminal tails can be modified by acetylation, which neutralizes the positive charge
Addition of an inducer stimulates production of an enzyme
- when a new carbon source is added to the growth medium of E coli. synthesis of the proteins needed to take up and metabolize it is induced - usually regulated at the level of transcription: mRNA encoding the the enzyme is induced - regulatory mechanisms can be pos or neg
Cis and Trans acting factors
- why are results of adding a plasmid copy different for lacl and lapP 1. lacl expressed from plasmid, makes repressor, can still bind to lacO *lacl= trans 2. lacP can bind to RNA pol, but can only initiate transcription for the lacZY if genes directly adjacent. cannot restore exp if lac genes on chromosome *lacP works in cis
Microbiome DNA analysis methods
-16 rRNA sequence: PCR amplify 16S rRNA gene--> sequence--> group sequence into OTU's; compare OTU sequence to databases--> identification of species and relative abundance of species within sample *OTU= operational taxonomic unit, similar to 16S sequence - total DNA seq: next gen seq of total DNA from microbiome sample--> filter host DNA sequences; compare to databases--> identify species; relative abundance of species within sample and genes and variants, polymorphisms and functional info
elongation (translation)
-Elongation factor EF-Tu, a G-protein, complexes in its GTP-bound form with a charged tRNA = tRNA-AA (a "ternary complex"), bringing charged amino acid to ribosome - tRNA-AA loaded into A site of the ribosome - EF-Tu is released upon GTP hydrolysis and recycled - if codon/anticodon interaction between tRNA and A site is correct, then GTP on EF-Tu is hydrolyzed, EF-Tu is released; -makes sure right amino acid is being put in; -first amino acid covalently linked through peptide bond to amino acid 2 - system shifts over, A site tRNA moves to P site, conformational change of A site, GTP hydrolyzed while EF-G fits into A site; ribosome moves forward; - aminoacyl-tRNA binds to A site
gene expression in eukaryotic cells
-Eukaryotic cells have a nucleus: DNA is in the nucleus; ribosomes are in the cytoplasm. -nuclear membrane shields the RNA from ribosomes until it is exported to the cytoplasm. -Transcription and translation are physically separated -Allows RNA processing - Trx and translation uncoupled
basic structure of a typical bacterial protein- coding gene
-ORF= protein coding sequence - TSS - promoter: RNA pol binds here, determines trx start, -10,-35 regions -transcription terminator -5' and 3' untranslated regions: regulation
gene expression in prokaryotic cells
-Prokaryotic cells lack a nucleus -Ribosomes can begin translating mRNAs as soon as they are made -Transcription and translation occur in rapid succession - circular chromosome
Initiation of Transcription
-alpha: enzyme assembly, promote interaction with regulatory proteins -Beta: catalysis -Beta': binds DNA catalysis -sigma: positions trx: binds to -10 and -30 regions of promoter; dissociates from RNA pol after elongation begins
how are specific amino acids attached to specific tRNA molecules
-amino-acyl tRNA synthetases *enzymes responsible for linking specific aa to the tRNA that recognize the codons for that specific amino acid
starch
-amylose: mostly unbranded, more resistant to digestion, not soluble in water - amylopectin: short, highly branched, higher levels of it result in sticky "glutinous" or "waxy" starch - highly branched starch has gelding properties used for food gels, adhesives, etc.
Wobble position
-deviations from standard W-C bp rules in codon-anticodon interactions at the 3' position of the codon
gene expression is controlled by the binding of regulatory proteins to regulatory regions of genes or operons
-neg regulation: repressor proteins bring to DNA, prevents RNA polymerase from binding and/or initiating transcription *in absence of repressor: transcription initiated--> repressor must be retrieved/ removed - positive regulation: activator proteins help recruit RNA polymerase to promoter, activate proteins help recruit RNA pol to promoter, activate transcription *In absence of activator low (basal) Level of transcription
PCR cycles
run for many cycles, each cycle has 3 steps 1. denature (90 degrees C) 2. anneal (60 degrees C) 3. Polymerization (72 degrees C)
transcription is activated by...
sequence specific activator proteins
genomics
study of whole genomes, including genes and their functions
Genome
the complete instructions for making an organism, consisting of all the genetic material in that organism's chromosomes
How is the PCR single tube made possible
through the thermostability of Taq polymerase... heat denaturation of the template does not inactive the enzyme
aminoacyle-tRNA synthetases charge tRNAs with specific amino acids
two steps 1. active aa (1-3) 2. transfer to tRNA (4-5)
Polymerase Chain Reaction
- 1980s - reiterative DNA synthesis in vitro using specific oligonucleotide primer pairs --> exponential amplification of a specific genomic DNA fragment - allows the isolation and amplification of specific DNA fragments from. large complex genome
dideoxynucleotide
- 2',3'-dideoxyribonucleotide - no 2'-OH on sugar (as expected for DNA) - no 3'-OH on sugar (unexpected!) -cannot form a phosphodiester bond; no 3'-OH substrate for DNA polymerase - once this base has been added by a DNA polymerase, no other nucleotides can be added to the chain
what does E. coli promoter look like
- an alignment of DNA seq in E.coli promoters reveal consensus seq. upstream of TSS (+1) - promoter strength correlates with the degree to which the promoter sequence matches the consensus
an E. coli genome undoes many genes
- very high density - 4.6 million bp - 4377 genes, many operons, no introns - both strands used
initiation of translation in prokaryotes
- 30s and 50s subunits not yet dissociated. kept separated by initiation factor 3 (IF3) - initiation factor 2 (IF2) delivers initiator tRNA charged with fMet to the P site (usually AUG) - established ORF *formyl group blocks reactive N atom of the amine group -IF2 ensures that any initiator tRNA-fMet enters p site - once tRNA-fMet in place 50s subunit of ribosome can bind - IF2 is a type of G protein: switches between GTP and GDP bound forms due to GTPase activity of the protein - when 50S su binds to 30s GTP is hydrolyzed to GPD and IF2 released - ribosome is properly positioned, ready to elongate
Histone proteins
- 4 histone proteins from a histone Octomer - 160 bp double-stranded DNA wraps around octomer - histone H1 binds on the outside of the "bead" - linker DNA connects to one "bead" to another - the histone fold handshake: H2A/H2B and H3/H4
bacterial vs eukaryotic regulation
- Bacterial: ground state is on, template for transcription is protein-free, activators enhance weak polymerase binding, repressors interfere with polymerase binding, polymerase holoenzyme binds protein-free DNA, promoters are DNA. - Eukaryotes: ground state is off, template for transcription is chromatin which is inaccessible to RNA polymerase, activators make chromatin accessible and/or promote the formation of a marked site on the DNA, repressors interfere with transcription by blocking activators or by making chromatin less accessible, RNA polymerase binds to protein-DNA complexes which mark the DNA, promoters are protein-DNA complexes
the major groove is not only accessible to DNA binding proteins but is also the most info-rich
- DNA binding proteins can make contact with different H-bond donors and acceptors - allows for "recognition" of specific binding sites
Prokaryotes and eukaryotes have related multi-subunit RNA polymerase structures
- E coli (prokaryotic) RNA pol * 6 su--> initiation * 5su--> elongation - Eukaryotic RNA pol * many subunits
gal80 in galactose
- Gal 7, Gal 10, Gal 1 genes in yeast are coordinately regulated by Gal 4 - Gal 4 proteins bind to yeast enhancers (upstream activation sequence (UAS) to activate transcription when galactose is present - in the absence of galactose, Gal 80 protein binds to Gal 4 activation domain *while Gal 4 binds to DNA, it can no longer activate transcription - in presence of galactose, galactose binds to Gal 3, which binds to Gal 80 in cytoplasm --> Gal 80 prevented from getting to nucleus *Gal 4's activation domain is now exposed to promote transcription initiation of Gal7, Gal 10, and Gal 1
what additional elements are required for transcription when the DNA is part of chromatin in vivo?
- Histone acetylation "Loosens" chromatin "ON" *complexes that mediate these interactions (and interactions of long-range enhancers and the promoter) - formation of transcription initiation complex (DeBoRaH) - chromatin remodeler to reposition nucleosomes (ATP-dependent)
tRNA anticodon: codon interactions
- IGC complementary to 3 Ala codons - some tRNAs must recognize multiple codons -Inosine (I) present in the anticodon loop of some tRNA molecules - (I)= deamination product of A, made after tRNA transcription - (I) bp with A,U, C in "wobble position" - an Ala tRNA with a 3'-CGI-5' anticodon can recognize 3 different codons *GCG cannot: has it's own tRNA
Techniques Based on in vitro DNA replication
- Polymerase Chain Reaction -DNA sequencing
How can a RNA (or DNA) sequence determine an amino acid sequence
- RNA linear code= 4 letters (A, C, G, U) --> translation - protein linear code is 20 letters (amino acids) - triplet code - codon= word in nucleotide language - genetic code is universal: enables genetic engineering
Overview of Transcription 2
- RNA pol binds to DNA template: at promoter - DNA helix unwound to form transcription bubble. rewound after transcribed - one strand is the template: for RNA synthesis - for any one gene, only one strand is the template -RNA is synthesized from rNTPs, 5' to 3' direction - form base pairs, complementary to the bases on DNA template - 5' end of transcript is displaced from template as polymerase moves
Elongation of RNA transcript
- RNA polymerization does not require a primer - 5' end of prokaryotic transcript has rNTP - reaction driven by hydrolysis of PPi
How do DNA and RNA differ?
- RNA: 2' has OH- ribose; 3' has OH - DNA: 2' has H-deoxyribose; 3' has OH - new nucleotides added at 3'OH - both in 5'-->3' direction -rNTPs and dNTPs are used as substrates during RNA and DNA synthesis, respectively
core promoter elements contain binding sites for GTFs
- TATA box: binds a subunit of TFIID called TBP
CRISPR/Cas9 genome editing
- a chemically synthesized RNA containing the sgRNA targets a specific DNA sequence - the CRISPR/Cas9 complex, containing the sgRNA, identifies its target - the sgRNA and target form a 20 nt RNA/DNA hybrid, opens the dsDNA - Cas9 cleaves both DNA strands with the unpaired DNA sequence - generates a ds DNA break - dsDNA break triggers DNA repair mechanism in cell - efficient and precise took for genome editing
CRISPR/Cas9
- a natural defense mechanism used by bacteria to protect against virus infection - Clustered regulatory interspaced short palindromic repeats - Cas9: CRISPR associated protein 9 nuclease - Cas nuclease targeted to specific DNA sequences by a single guide RNA (sgRNA or gRNA), encoded by repeat sequences in bacterial CRISP locus
catabolite activator protein (CAP)
- activates by recruiting RNA polymerase by binding C- terminal domain of the alpha subunit
combination of cooperatively bound transcription activators regulate eukaryotic cells
- activation requires multiple factors, some of which bind to DNA cooperatively - different genes require different combinations of activators - activation of the same gene at different times and places also involves different combinations of activators
The Ribosome is a ribozyme
- an RNA-only active site- no proteins within 20 angstroms of the site of peptide bond formation, implicating rRNA as a ribozyme
transfer RNA=tRNA
- an adapter molecule: brings AAs corresponding to each codon ribosome - small; 73-93 nt - conserved clover leaf 2-D structure - presence of modified bases include inosine (I) - amino acid attachment at the 3' end of the tRNA at the end of the CCA-3' stem - anticodon loop interacts with complementary codons on the mRNA by base-pairing
nucleosomes and chromatin
- avg human chromosome: 5 cm long - nucleus of a human cell: 5-10 microns *need to pack to reduced DNA volume - nucleosomes: DNA (about 160bp) is wrapped around a protein core made of 8 histone molecules - adjacent nucleosomes coil into a chromatin fiber - think of chromatin as a compact structure that needs to be made accessible for transcription, replication, and DNA repair
Sanger dideoxy sequencing technology
- based on in vitro DNA synthesis from a fixed site on a purified DNA template 1. denature 2. add one oligonucleotide primer complementary to a short stretch of one strand 3. add DNA polymerase and dNTPs (BUT add dideoxy... then any time there is a C, can put dideoxy C (ddCTP); chain terminating 4. do it again--> synthesis stops (at G-C pairs) * will end at C 5. separate molecules using gel electrophoresis
sigma subunit of E. coli RNA polymerase
- binds to the -10 and -35 consensus seq elements - the subunit of the holoenzyme contacts -10 and -35 regions - fits in major groove
redundancy of the genetic code
- can be read out by a combo of wobble bp - presence of multiple ind. RNA molecules with different anticodons carrying the same aa - ex: Alanine: 4 codons, not 4 tRNAs *only 2
an aminocyl-tRNA linkage
- carboxyl group of aa is linked to 3'OH of tRNA - corresponds to CCA at 3'end - charged tRNA
sickle cell disease
- caused by mutations in the B-globin gene -cause by a single nucleotide mutation (CTC to CAC) in exon 1, changes Glu at codon 6 to Val - results in change in shape--> clumping -gene editing patient- derived hematopoietic stem cells followed by autologous transplantation could potentially be used to cure B-haemoglobin opathies
activation of the lac operon in the presence of lactose but low glucose
- cell does not want to activate expression of lac operon if there is plenty of glucose around - glucose is a better carbon source - what do you think cell might use as an inducer to convert CAP into an active DNA binding protein - an indicator of energy status--> cAMP - cAMP high when glucose low
how does the ribosome know where to start
- choice of the AUG initiator codon in prokaryotes depends on bp between 16s rRNA and the shine-dalgano sequence on the mRNA - optimal spacing is 5nt, but can be <13 nt
N terminal tails
- conversed and are targets for covalent modules that regulate chromatin structure and provide binding sites for proteins - the N- terminal tails of histones are targets of extensive modification (mostly R and K)
transcription and translation are coupled in prokaryotes
- coupled transcription and translation in prokaryotes - "polysomers" - multiple ribosomes translating a single mRNA transcript
Illumina sequencing
- currently used in DNA sequencing - sequencing by synthesis -attach adapters to create a sequencing library of selected 200-300 bp fragments
DNA sequencing
- determination of the linear order of nucleotides *Sanger vs next generation sequencing
tRNA and tRNA synthetase recognition
- each amino-acyl-tRNA synthetase has binding pockets for specific AA and its cognate tRNA - "discharging" error rate about 10^-4 to 10^-5
Termination of translation in prokaryotes
- elongation cycle continues until a stop codon is encountered - termination mediated by a protein Release Factors (RF) that interact with stop codons in the A site - RF structurally resemble tRNA - probably allow water access to ester bond between AA and tRNA in P site - ribosome dissociates ,tRNAs and completed polypeptide are released
Living organisms respond to their environment
- ex: hiker who sees bear and her cub - ex: e.coli induce lactose metabolizing operon in presence of lactose
A typical eukaryotic protein coding gene
- exons and introns - one ORF per trx ( no operons) -5' and 3' untranslated regions (UTR) (poly-A signal) - promoter and upstream regulator regions -transcription start site (+1) - Trx termination
dye-terminator Sanger sequencing technology
- fluorescent labels on ddNTPs: four diff flours... one type attached to each ddNTP type - can determine which one ends on (A,T,C,G)
How does Gal4 activate transcription in yeast
- galactose: gal4 inhibited by gal80 bound to activation domain + galactose: galactose/gal3 binds and sequesters gal80 in the cytoplasm *gal4 activation domain binds a histone acetyltransferase coactivator complex (SAGA) and mediator which recruit GTF's and PolII to promoter
Applications of PCR
- genetic engineering -genetic variation detection - evolutionary DNA investigations
DNa/Histone interactions
- histones interact with DNA minor groove; A/T base pairs are favored - implication: sequence-specific positioning of nucleosome on DNA
Importance of Cis and Trans-acting factors
- importan for understanding gene regulation - regulatory proteins can be encoded at genetic loci physical distant from the genes that they regulate -DNA regulatory elements need to be physical close to the genes whose transcription they regulate --> in prokaryotes usually direct adjacent to genes they regulate --> regulatory elements can function at a greater distance in eukaryotes
Application for CRISPR/Cas9 cont.
- imprecise intertions or deletions from (NHEJ) --> often loss of function - precise changes in nucleotide sequence through homology directer repair (HDR) - desired new sequence (donor DNA) must be provided -ex: correction of sickle cell mutation in a haemoglobin gene
Sanger dideoxy sequencing
- in vitro DNA synthesis on a purified DNA template from a fixed site, which is determined by the position of a single oligonucleotide primer - dna synthesis reactions "poisoned" by chain terminating "dideoxy" nucleotides, each associated with a different fluorescent dye - based on DNA polymerization and chain termination
lac repressor and many other bacterial DNA binding proteins bind to DNA using helix-turn-helix DNA binding domains
- lac repressor binds to operator as a dimer R-groups on one side of helix - makes contact with major groove atoms
regulation of gene expression
- prokaryotes and other single-celled organisms respond to changes in their environment through altering gene expression * new carbon source, nitrogen, etc. * change in light, temp, stress conditions * presence of host cell - prokaryotes are very efficient: only express genes, produce enzymes when they are needed - couple expression of genes to a sensing system - in multicellular organisms, changes in gene expression allow for acclimation to different environmental conditions and for formation of different cell types (development)
Mammalian Pol 2 gene
- promoter- sequences adjacent to transcription start point; determines where transcription starts - the set of sequences required to recruit general transcription factors and RNA pol to non-chromatin DNA in a test tube= core promoter - promoter- core promoter plus nearby elements. minimal sequence required to recruit DNA pol to transcription start site in cell - enhancer elements: enhancers work from far away to increase transcription. They are needed for maximal promoter activity, and often involved in cell-type specific expression ex: sonic hedge hog gene
alternative prokaryotic promoter sequence
- promoters of different classes of genes are recognized by alternative sigma subunits
Ribosomes read the instructions for protein synthesis on mRNA
- read the instructions for proteins synthesis from mRNA molecules - bind mRNA and tRA-AA - positions them properly - three tRNA binding sites: A: aminoacyl/ acceptor P: peptiyl E: exit - polarity *mRNa read 5' --> 3' * aa chain is N-term to C-term
culture independent methods for describing microbial communities
- rely on sequencing DNA of organisms within a community -metagenomics: study of genetic material recovered directly from environmental sample - two approaches: 1. targeted metagenomics: sequencing single genetic locus within community DNA 2. shotgun metagenomics: sequence community DNA in unbiased manner - allows analysis of various functions in community
The 16S rRNA gene acts as a molecular fingerprint that can be used to identify different bacteria
- ribosomes in prokaryotic and eukaryotic cells are similar, but not identical - eukaryotic ribosomes do not have a 16S rRNA molecule - the presence of both conserved and variable regions is critical
how do we learn about genomes
- sequence and analyze entire genomes * humans have 25,000 genomes, 3 x 10^9 bp - rough correlation between gene number and complexity of the genome - examining the genes, their predicted functions
elongation
- sigma dissociates after initiation - RNA pol core enzyme carries out synthesis - transcription bubble: about 17 bp unwound - RNA-DNA hybrid: about 8-10 bp - error rate 10^-4 - 10^-5 - trx rate: 50 nucleotides/ sec
PCR steps
- start with 1 molecule of double stranded DNA (template DNA) - heat denature at 95 degrees C: single stranded separation - primers at lower T anneal to their complementary sequence each strand--> most olives used as PCR primers are DNA 20-25 nt in length - DNA polymerization (taq pol and dNTPs at 70 degrees C) *taq pol requires a 3'-OH, template, and polymerizes DNA in a 5'-3' direction * 1 cycle of denaturation--> primer annealing--> polymerization= 2 double stranded DNA molecules
termination of transcription: Intrinsic mechanisms
- termination sequence: G-C rich region followed by string of A's - after transcription through 3'UTR RNA forms haripin loop - triggers release of completed transcript and RNA polymerase from DNA
How is a genetic code read
- the aa's themselves cannot read it - read by tRNA molecules; aminoacly-tRNA molecules
Jacob and Monod used genetics to study lac operon
- tools: *wild type E coil: lactose induces expression of B gal, lac permease * 2 types of ecoli mutants 1. in structural genes: lacY (no permease), lac 2 ( no B gal) 2. regulatory mutants: lacP, lacO, lacl * partial diploids: 2nd copy of small DNA segment on F' plasmid - phenotypes of lac operon mutants *lac Z, lac Y: disrupted proteins, no activity * lacP: promoter impaired, less or no activity * lacl: lacZYA genes not regulated by lac, const, expressed *lacO: lazZYA not regulated by lact; constitutively expressed (repressor cannot bind)
Features of the Genetic Code
- triplet code: 64 different 3 nucleotide "codons" - non-overlapping - three diff "reading frames" on each strand *move over by 1... then another - open reading frame * do not encounter any stops - redundancy - only 20 aa to be encoded by 64 possible codons - synonymous codons specific the same aa - punctuation: start = AUG (met); stop= UAA,UGA,UAG - ORF: is a long string of amino-acid specifying codons that begins with an AUG codon and is not interrupted by stop codons
Ribosome composition in prokaryotes
- two subunits: large 50S and small 30S - each subunit contains both ribosomal RNA (rRNA) and many proteins - 50S = 23S rRNA + 5S rRNA + 31 proteins - 30S = 16S rRNA +21 proteins - eukaryotic ribosomes are similar but not identical - proteins are stabilizers. poke into crevices
Partial diploids allowed J&M to test whether regulatory elements of lac operons worked in cis or trans
- use of partial diploids a common genetic approach - extra DNA fragments carried on a plasmid - two copies of part of the genome - allow to ask if 2nd copy of DNA on a plasmid can restore wild type phenotype to a mutant -dom/recessive relationship? - if genetic element must be physically adjacent to lac operon --> in "cis" - if genetic element can be located on a separate piece of DNA --> in "trans"
CRISPR/Cas9 used to edit B-haemoglobin gene in human haemopoietic stem cells
- used CRISPR/Cas9 to correct the Glu6Val mutation in SCD in HSPC- from the SCD patients - created therapeutic rAAV6 viral donor construct (corrective donor) designed to revert the mutation -gRNA directs cleavage in Exon 1 - tested whether the HBB-edited HSC's still able to differentiate into RBCs that could express haemoglobin (Hb) - observed presence of matured differentiated erythrocytes that expressed Hb - targeting HSC with corrective WT donor --> 50% of GluVAL (Hb) alleles to wild-type (HbA) alleles--> works! - still needs optimization: off-target mutations can occur
Next (Current) and Next Next Gen. DNA sequencing
- very fast moving - massively parallel seq: millions to billions of reactions happening and being recorded at the same time by a single sequencer - current: direct detection of DNA bases s they are incorporated into a growing DNA strand - newest: direct detection of DNA bases without replication as they pass by some sensor
Application for CRISPR/Cas9: Modification of starch content in corn
-objective: generate corn with higher levels of the amylopectin form of starch= "waxy" - starch isolated from waxy corn kernels has unique applications in food and industry (adhesives) - starch: polymers of glucose *branching patten impacts the properties of starch - the waxy (wx1) mutation of corn: results in modified starch content *normal no.2 yellow dent corn has 75% amylopectin *the (wx1) mutation from the 1900s contain >97% amylopectin * breeders would like to move trait into many different corn lines - in 2016, announced product from CRISPR-Cas9: next-gen of waxy corn hybrids - applications of CRISPR/Cas9 *2 gRNAs to cut in xxl locus about 4kb apart *non-homologous end joining repairs break *generated a 4kb selection of the WX1 coding sequence--> loss of function
In a gene
-promoter: determines where transcription starts -regulatory sequence: when, how much - coding sequence: Open Reading Frame, ORF, if gene encodes protein - transcription terminator: where transcription should end
phenotypic characterization of CRISP- waxy plants
-worked well - starch from CRISPR identical to conventional-bred (TI-WX) - faster than traditional
Histone tail interacting protein classifications
-writers: covalent modification of histone aa - erasers: restore modified histone aa to to unmodified form - readers: bind modified histone aa ex: * writers: histone acetyltransferase activates transcription * erasers: histone deacetylase; represses transcription
roles of TFIIH during transcription
1. DNA "melting" catalyzed by a TFIIH helicase 2. Phosphorylation of carboxyl terminal domain (CTDs) of RNA pol II by a TFIIH kinase
activators have two separable domains
1. DNA binding 2. activation - if DNA binding domains are swapped out for one another, activation still works
targets of activation domains are recruited by the promoter by protein-protein interactions among these targets (co-activators)
1. GTF a) TFIID-major one (TBP and TAFs) b) mediator complex- associated with RNAP 2. histone code writers (HATS) 3. chromatin remodeling complexes
3 phases of transcription
1. Initiation: RNA pol recognizes and binds to promoter sequence, determines starting point for transcription (+1) 2. Elongation: RNA pol monitors binding of rNTP to next base on template. If match, catalyzes bond formation 3. Termination: RNA pol pauses and dissociated from the template. usually at designated termination site
sequential recruitment of GTF and assembly of the eukaryotic RNA pol II PIC
1. TFIID binds to core promoter elements 2. TFIIB binds to TFIID/DNA 3. Pol II RNA pol binds to TFIIB/TFIID/DNA 4. TFIIH binds to complex 5. TFIIH opens DNA phosphorylates CTD to allow initiation * phosphorylation of c-terminal domain of RNA POL II helps open transcription bubble
How to figure out "who" is there
1. examine species under microscope 2. culture (when possible) 3. culture-independent technologies -isolate total DNA from environmental samples and sequence
genetic approaches for investigating gene function
1. intro of extra or altered versions of genes into the genome (transgenic approach) 2. reverse genetics and genome editing
acetylation of lysine in histone tails
1. loosens chromatin structure by relieving nucleosome-nucleosome interactions and histone-DNA interactions 2. it provides binding sites for proteins that facilitate activation
types of prokaryotic RNA
1. messenger RNA (mRNA): encode proteins 2. transfer RNA (tRNA): bring correct amino acid to mRNA 3. ribosomal RNA (rRNA): major component of ribosomes 4. small RNA (sRNA): regulate gene expression - each made by transcription of DNA using the same RNA polymerase
reverse genetics and genome editing
1. reducing expression of a gene -RNA 2. introducing imprecise changes in a gene -insertion or deletions - can result in loss of functions 3. introducing precise changes: e.g. specific nucleotide (homology directed repair= HDR) - CRISPR/Cas 9
Genome Analysis
1. sequencing entire genome to determine its DNA sequence 2. mapping genomes to specific locations on chromosome 3. examine gene expression pattern 4. determine the function of genes - reverse genetics 5. investigating interactions between genes
Features of transcription in eukaryotes
1. spatial separation of transcription and translation in eukaryotes; 2.more complex transcriptional regulation: -three RNA polymerases -GTFs -cis-acting elements (DNA regulatory sequences that bind proteins) are more varied and can be positioned in different configurations relative to the coding sequence 3. extensive processing of primary transcript destined to become mRNA in eukaryotes
PCR: Single Tube
1. template DNA 2. DNA polymerase 3. dNTP's (dATP, dCTP,dGTP, dTTP) 4. Buffer 5. a pair of single-stranded oligonucleotide primers that hybridize to a specific genomic locus (position) - orientation 5'-3' 3'-5'
how many transcription factors does protein-protein interaction allow to bind cooperatively to DNA
2 - cooperative= each binds better to its site on the DNA in presence of each other
PCR amplification
proceeds through successive rounds by raising and lowering T (using a PCR machine) - 2^n double stranded DNA segments (n=# of cycles)
exponential amplification
process is repeated, and the region of interest is amplified exponentially
linear amplification
relationship between input and output is proportional, so that the low-intensities are amplified the same as the high intensities
Central Dogma
DNA -> RNA -> Protein
Neg control
repression of the lac operon in the absence of lactose (inducer) - lac represor proteins bind to lacO and prevent RNA pol from binding to promoter - inhibits transcription of lac operon - de-repression of the lac operon in the presence of lactose (inducer) * polycistronic mRNA translated
mediator
not required for basal transcription in vitro but it also considered to be a GTF 1. required for transcription of most cells 2. interacts with RNA polymerase, other GTFs, and with activator proteins bound to far-away enhancers
In the absence of chromatin, what transcriptional machinery do eukaryotes require
General transcription factor (GTF)
GTFs and RNA pol assemble at the core promoter to form...
Pre-initiation complex (PIC) which is required for basal transcription in a test tube = TFIIB =TFIID =TFIIH
regulation of transcription in initiation
a: basal expression: RNA polymerase alone (no activator or repressor, RNA pol) binds occasionally to promoter. expression at low (basal) level b: negative regulation: binding of a repressor blocks binding of RNA polymerase, inhibits transcription c: positive regulation: binding of an activator recruits RNA polymerase. expression is activated, high levels of expression - ecoli. lac operon regulated by an activator and a repressor working in this way
Total molecular weight of GTF
about 1 megadalton
Molecular definition of a gene
all information needed to synthesize RNA or protein
B-galactosidase
breaks down lactose into glucose and galactose
gene expression
changes in single-celled organisms allows the organism to adapt to different environmental conditions - changes in multi-cellular eukaryotic organisms allows formation of different cell types in addition to allowing adaptation to different environmental conditions
Microbial community composition of different body location in a healthy human
community composition varies a lot with location
Genetic Definition of a gene
everything required to add back to a mutant to restore function of the gene (complementation)
DNA
genetic material; encodes genes
In vitro
in test tube
Allosteric effectors
inducers - bind to regulatory proteins, influence DNA binding - inactivation of repressor: inducers bind to repressor protein at a 2nd site, change ability of protein to bind to DNA - activation of activator: inducer vs inducer present
DNA replacement
involves homologous recombination: exchange of genetic info between DNA molecules that have high levels of homology (seq. identity) - occurs frequently in bacteria, yeast -also occurs in mice, other mammals - has been used to "knockout" every predicted ORF in yeast, also in mice
