Gene regulation in bacteria
Lvl of gene expression varies under diff conds
Majority genes are regulated--so proteins they encode can be prod at proper times & in proper amts
Cells are efficient b/c proteins encoded by regulated genes are only present in cell when needed
Constitutive genes
Unregulated genes that essentially have constant lvls of expression in all conds over time
Usually encode proteins continually needed for survival of bacterium
Examples of regulated processes
Metabolism--certain enzymes needed for bacteria to metabolize particular sugars
Response to environmental stress--certain proteins help bacteria survive environmental stress, like osmotic shock or heat shock
Cell division--some proteins needed for this pr
Transcriptional regulation
Turning genes "on" or "off" usually just refers to decr or incr rate of transcription; usually occurs at this lvl by influencing rate of RNA synthesis
Usually involves actions of regulatory proteins that can bind to DNA & affect rate of transcription of 1
Negative control
Gene regulation controlled by repressor--regulatory protein that binds to DNA & inhibits/decr rate of transcription
Positive control
Gene regulation controlled by activator--regulatory protein that binds to DNA & stimulates/incr rate of transcription
Effectors
Do NOT bind directly to DNA
Small mlcs that binds to activator or repressor; causes conformational change in regulatory protein, influencing whether or not it can bind to DNA
Regulatory proteins that effectors bind to must have 2 functional domains (bindi
Inducers
Effector that causes transcription to incr; does so in 2 ways...
1.) Bind to repressor protein & prevent it from binding to DNA (inhibit repressor)
2.) Bind to activator protein & cause it to bind to DNA (stimulate activator)
Genes regulated like this are
Corepressors
Effector that causes transcription to decr by binding to repressor & causing it to bind to DNA (stimulates repressor)
Genes regulated like this are called repressible genes
Inhibitors
Effector that causes transcription to decr by binding to activator & preventing it from binding to DNA (inhibits activator)
Genes regulated like this are called repressible genes
Enzyme adaptation
Observation that a particular enzyme appears w/in living cell only after the cell has been exposed to the substrate for that enzyme (in other words, the phenomenon that certain enzymes are only present when the cell needs them)
Jacob & Monod
Studied enzyme adaptation by examining lactose metabolism in E. coli
Experiments showed?????
operon
Group of 2 or more genes under transcriptional control of a single promoter
Encode for a polycistronic RNA--RNA that contains sequences for 2 or more genes
Biological advantage--expression of genes w/common functional goal can occur as a single unit
1 pro
lac operon
Transcriptional unit involved in utilization of lactose
Contains: CAP site, lacP, lacO, lacZ, lacY, lacA, & terminator
CAP site
Short DNA sequence recognized by Catabolite Activator Protein (CAP)
lacP
Promoter site
lacO
Operator site (or just operator)--short DNA sequence that provides binding site for repressor protein
lacZ
Structural gene that encodes beta-galactosidase--enzyme that cleaves lactose into galactose & glucose; also converts small amt of lactose into allolactose
lacY
Structural gene that encodes lactose permease--membrane protein needed to actively transport lactose into cytoplasm of bacteria
lacA
Structural gene that encodes galactoside transacetylase--covalently modifies lactose
(don't need to know??)
lac operon regulation
lac operon is regulated by lacI gene which has its own promoter (i promoter)--this gene encodes mRNA that is translated into lac repressor protein & ????
Describe the 1st mechanism by which the lac operon is regulated
Inducible & under neg control
lac repressor protein binds to lac operator site--> inhibiting RNA polymerase from transcribing lacZ, lacY, & lacA (structural genes)
Reversible process
In absence of allolactose, lac repressor is bound to operator most of th
lac repressor structure
1 protein domain that binds to DNA
1 protein domain is a homotetramer--made up of 4 identical subunits, each w/a single binding site for allolactose
DOES EACH SUBUNIT ALSO CONTAIN ACTIVE SITE TO BIND TO DNA or IS THE HOMOTETRAMER STRUCTURE JUST FOR THE AL
lac repressor function
Important for regulation of lac operon
Only small amt needed to repress lac operon
Allolactose
Inducer
Binds to repressor; changes repressor's shape in a way that inhibits it from binding to operator site
Referred to as allosteric regulation
Effect of allolactose on operon
Operon is induced (incr transcription rate)
If present--> transcription occurs b/c it inhibits the repressor
If absent--> repressor binds to DNA & transcription cannot occur
Allosteric regulation
lac repressor protein is also an allosteric protein (has allosteric site & active site)
-->Allosteric site = where effector mlc (i.e. allolactose) binds to repressor
-->Active site = where repressor binds to DNA
What determines the likelihood that allolactose will bind to the repressor?
Concentration of allolactose (lac repressor has measurable affinity for allolactose)
What happens to the concentration of allolactose during induction of the operon?
Bacterium is exposed to lactose--> small amt of lactose moved into cytoplasm (via lactose permease)--> converted into allolactose (via beta-galactosidase)--> cytoplasmic lvl of allolactose gradually incr, eventually reaching affinity for lac repressor; th
As the cell uses lactose, what happens to allolactose levels and what is the result?
Incr transcription of lacZ, lacY, & lacA result in greater prod of proteins that break down lactose--> allolactose concentration decr, eventually below affinity for lac repressor; thus allolactose unlikely to be bound to lac repressor (attached to operato
Trans-effect
Genetic regulation that can occur even though 2 DNA segments are not physically adjacent
Mediated by genes that encode regulatory proteins
Ex: ?????
Cis-effect
Genetic regulation that occurs when 2 DNA segments are physically adjacent
Mediated by DNA sequences that are bound by regulatory proteins
Ex:??????
Describe the 2nd mechanism by which the lac operon is regulated
Catabolite repression--presence of glucose represses lac operon; glucose is a catabolite
Catabolite--substance broken down inside the cell
Diauxic growth
Sequential use of 2 sugars (common among bacteria)
Bacteria metabolize glucose first
Advantage--greater efficiency b/c bacteria doesn't have to express all of the genes needed for both glucose & lactose metabolism
CAP
Catabolite Activator Protein--activator protein that binds to CAP site
Structure--made up of 2 subunits, each of which can bind 1 mlc of cAMP
Active if cAMP is bound to it
Inactive if cAMP is not bound to it
Cyclic AMP (cAMP)
Inducer that binds to CAP (the activator) causing cAMP-CAP complex to bind to CAP site, near lac promoter--> stimulating transcription
cAMP-CAP complex
Structure--2 mlcs of cAMP bound to CAP (1 cAMP mlc for each subunit)
Function--bind to CAP site to stimulate ability of RNA polymerase to begin transcription
Lactose & no glucose
Repressor is inactive b/c lactose present
cAMP-CAP complex is active b/c high cAMP--> enhances binding of RNA polymerase to promoter
Result--high rate of transcription
Lactose & glucose
Repressor is inactive b/c lactose present
cAMP-CAP complex is inactive b/c low cAMP
Result--low transcription rate
No lactose or glucose
Repressor is active b/c no lactose present
cAMP-CAP complex is inactive b/c high cAMP
Result--very low transcription rate
Glucose & no lactose
Repressor is active b/c no lactose present
cAMP-CAP complex is inactive b/c low cAMP
Result--very low transcription rate
How many operator sites does lac operon contain? What is the location of each relative to other important sites on the lac operon?
3 operator sites
O3 & O1 are upstream from lacZ; O3 is also upstream from CAP site & lac promoter (lacP); O1 is at lacO--downstream from CAP site & lacP
O2 is downstream from lacZ; downstream from CAP site & lacP, btwn lacZ & lacY
General structure...
i p
DNA loop formation
...
Under what conditions does the loop form?
...
Why would transcription not be taking place even though the cAMP-CAP complex is binding to the CAP site?
????
ara operon
Found in E. coli (bacteria); involved in sugar (arabinose) metabolism; arabinose is a constituent of cell walls in certain plants
Contains 3 structural genes (araB, araA, araD) that encode a polycistronic mRNA for 3 the 3 enzymes involved in metabolizing
ara operon regulation
AraC protein may negatively or positively control transcription depending on whether or not arabinose is present
AraC protein
Encoded by araC gene (adjacent to ara operon)
Binds to araI, araO1, & araO2 sites
How does AraC act in presence of arabinose?
POSITIVE CONTROL--AraC protein = activator
Arabinose binds to AraC protein--> interaction btwn AraC proteins at araO2 & araI sites is broken--> opens DNA loop
-->Arabinose = inducer
A 2nd AraC protein binds at araI site, forming AraC dimer--> activates tr
How does AraC act in absence of arabinose?
NEGATIVE CONTROL--AraC protein = repressor
AraC protein dimer binds to araO1--inhibits transcription of araC gene
-->Acting as repressor of araC gene
-->Keeps AraC protein lvls fairly low
AraC monomers bind to araO2 & araI--causing DNA loop--> RNA polymer
What structures are in both the lac and ara operon?
1 promoter for the entire operon, CAP site, & an important adjacent gene w/its own promoter (lacI; AraC)
Relationship between glucose levels and cAMP levels?
High glucose concentration = low cAMP lvls; low glucose concentration = high cAMP lvls
Glucose transported into cell-->causes signaling pathway that inhibits adenylyl cyclase (enzyme needed for cAMP synthesis)--> cAMP concentration decreases
trp operon
Contains structural genes: trpE, trpD, trpC, trpB, & trpA that encode enzymes needed for biosynthesis of tryptophan
trpR & trpL genes involved in regulating trp operon in 2 diff ways
trpL & trpR
trpR gene encodes trp repressor protein
trpL gene mediates attenuation
trpE, trpD, trpC, trpB, trpA
Genes that encode enzymes for biosynthesis of tryptophan
What happens when tryptophan levels are low?
trp repressor CANNOT bind to operator site--> RNA polymerase CAN transcribe trp operon--> genes needed for synthesis of tryptophan are expressed
What happens when tryptophan levels are high?
Tryptophan = corepressor--binds to trp repressor--> changes its shape so that it can bind to trp operator site--> inhibiting transcription of trp operon
Attenuation
Controlled by trpL--encodes short peptide called leader peptide which functions in attenutation
What do inducible operons usually encode?
Catabolic genes
Ex: lac & ara operons (inducible systems)
Purpose--allows bacterium to express appropriate genes only when they are needed to catabolize (in this case sugars)
What do repressible operons usually encode?
Anabolic genes
Ex: trp operon (repressible system)
Purpose--provides bacterium w/way to prevent overprod of a biosynthetic pathway
Majority of gene regulation in bacteria occurs at what lvl?
Transcriptional
Translational regulation
Usually aimed at preventing initiation stage
Involves proteins that influence the ability of the ribosome to form a polypeptide
Translational regulatory proteins & translational repressors
Recognize sequences w/in mRNA; usually inhibit translation--> called translational repressors, which bind to mRNA & inhibit translational initiation in 1 of 2 ways...
1.) Repressor binds in vicinity of Shine-Dalgarno &/or start codon--> sterically blocks
Antisense RNA
RNA strand that is complementary to mRNA--forms double stranded structure via H-bonding btwn complementary regions, so that mRNA does NOT get translated
Ex: Osmoregulation...
At high osmolarity, the ompF gene "turns off" b/c micF gene is transcribed & its
Posttranslational regulation
Functional control of proteins that are already present in the cell; can activate or inhibit protein function
Advantage--occurs relatively faster (in just seconds); whereas transcriptional & translational regulation can take minutes or even hours to take
Feedback inhibition
...
Posttranslational covalent modification
Structure of the protein is changed (covalently modified)
Irreversible & reversible modifications
Irreversible modifications
Involved primarily in assembly & construction of a FUNCTIONAL protein
Alterations include: proteolytic processing (breaking down proteins into smaller polypeptides or amino acids); disulfide bond formation (like in DNA); attachment of prosthetic groups (l
Reversible modifications
Transiently affect function of a protein
Alterations include: phosphorylation (--PO4); acetylation (--COCH3); methylation (--CH3)
Phage lambda
Phage lambda can follow lytic or lysogenic life cycle (depends on cellular conds) after binding to surface of bacterium & injecting its genetic material into bacterial cytoplasm
Lytic cycle
Bacteriophage's genetic material instructs it to make many copies of phage's genetic material & coat proteins, which assemble to make new phages; bacterial host is lysed (bursts); newly made phages are released into environment
Lysogenic cycle
Lambda phage acts as temperate phage--doesn't prod new phages or kill bacterial host (dormant state)
Phage integrates its genetic material into bacterial chromosome--this integrated phage DNA = prophage
Prophage may exist in dormancy for a while (multiple
What controls lysogenic cycle?
Lamda repressor protein--binds to O(subscript--L) & O(subscript--R) (operator sites); binding to O(subscript--R) inhibits expression of genes needed for lytic cycle
What controls lytic cycle?
Cro protein--binds to O(subscript--L) & O(subscript--R) (operator sites) & has several effects...DO WE NEED TO KNOW THE SPECIFICS OR JUST THAT GENES FOR LYSOGENIC CYCLE ARE NOT TRANSCRIBED, BUT THOSE FOR LYTIC ARE????
What does the O(subscript-R) region do?
Provides genetic switch btwn lytic & lysogenic cycles
During lytic cycle--> cro protein controls switch
During lysogenic cycle--> lambda repressor controls switch
How many operators does O(subscript-R) region contain?
3 operators: O(subscript--R1), O(--R2), & O(--R3)
Lambda repressor & cro protein can bind to all 3 sites
Determines switch btwn lytic & lysogenic cycles
Effects of lambda repressor
Lambda repressor turns off P(--R) and turns on P(--RM), sending cycle to lysogenic side
Effects of cro protein
Early in lytic cycle--> cro protein turns off P(--RM), which switches off the lysogenic cycle, & turns on P(--R)
Later--> cro protein occupies all three O(--R) sites--> switching off P(--R), which is not needed in later stages of lytic cycle
What happens at the beginning of both reproductive cycles?
After phage lambda's DNA (linear) is injected into bacterium, its ends covalently attach, forming circular DNA
Early promoters & operators are transcribed soon after infection & play role in initiating competition btwn lysogenic & lytic cycles
If lysogenic cycle prevails, which gene is turned on?
Integrase (int) gene--encodes enzyme that integrates lambda DNA into bacterial chromo
(Genes on the left side of the diagram)
If lytic cycle prevails, what happens?
Genes necessary for synthesis of new phages are turned on--encode replication proteins, coat proteins, coat assembly proteins, proteins to package DNA into phage head, & lysis causing enzymes (to lyse host)
(Genes on the right side & bottom of diagram)
EUKARYOTIC GENE REGULATION
...
Transcription factors
Proteins that influence ability of RNA polymerase to transcribe a gene; either by regulating binding of transcriptional apparatus to core promoter &/or controlling the switch form initiation to elongation
General transcription factors
Regulatory transcrip
General transcription factors
Needed for binding RNA polymerase to core promoter & its progression to elongation stage (in other words needed for basal lvl of transcription)
Regulatory transcription factors
Regulate rate of transcription in specific (targeted) genes
Control/Regulatory elements
Cis-acting elements located near core promoter
Analogous to operator sites found near bacterial promoters
**Remember cis-acting elements = DNA sequences that trans-acting (in this case regulatory transcription factors) factors bind to cis-element
Activator & enhancer
Activator (the regulatory transcription factor) binds to enhancer (the DNA sequence near core promoter)--> stimulates transcription
Repressor & silencer
Repressor (the regulatory transcription factor) binds to silencer (the DNA sequence near core promoter)--> inhibits transcription
What are the trans-acting factors?
Activator & repressor
What are the cis-acting elements?
Enhancer & silencer
Combinatorial control
Phenomenon found in most euk (esp in multicellular species), in which genes are regulated my many factors
What are the 5 common factors that contribute to combinatorial control at the level of transcription?
1.) 1 or more activator proteins may stimulate ability of RNA polymerase to initiate transcription
2.) 1 or more repressor proteins may inhibit ability of RNA polymerase to initiate transcription
3.) Function of activators & repressors may be modulated in
Statement
There are diff families of evolutionarily related transcription factors; mlcular structure of transcription factor proteins that allows them to bind to DNA has become an intense area of research
Domains
Regions of transcription factor proteins that have specific functions
Ex: 1 domain of a transcription factor may DNA-binding function, & another domain on the same transcription factor may be binding site for effector mlc
Motif
Domain that has similar structure in many diff proteins
What secondary protein structure is frequently found in regulatory transcription factors?
Alpha helix
What's important about the alpha helix structure?
It has the proper width to bind into major groove of DNA double helix
Recognition helix recognizes & makes contact w/base sequence along major groove of DNA; H-bonding btwn alpha helix & nucleotide bases holds it together
Helix-turn-helix motif
Helix-loop
Homodimer
Formed when 2 identical transcription factor proteins come together
Heterodimer
Formed when 2 diff transcription factor proteins come together
Dimerization
Dimerization of transcription factors can be an important way to modulate their function
Considering regulatory transcription factors do not bind directly to RNA polymerase, how do they exert their effects on it?
Via TFIID & mediator
TFIID
General transcription factor that binds to TATA box--needed to recruit RNA polymerase II to core promoter
Regulatory transcription factors & TFIID
Regulatory transcription factors bind to regulatory element--> influences function of TFIID
Activators either help recruit TFIID to TATA box or enhance ability of TFIID to bind RNA polymerase II
-->Activators may exert effects by interacting w/coactivator
Mediator
Protein complex that mediates interaction btwn regulatory protein factors & RNA polymerase II
Controls ability of RNA polymerase II to progress to elongation stage
Regulatory transcription factors & mediator
Activator binds to distant enhancer--> activator interacts w/mediator to enable RNA polymerase to form preinitiation complex-->allowing it to move on to elongation
Repressor binds to distant silencer--> repressor interacts w/mediator to inhibit RNA polyme
Why is it necessary for the functions of regulatory proteins to be modulated?
Just like genes must be regulated, so must the regulatory proteins, b/c...
The genes they control must be turned on at the right time, in correct cell type, & under right conds
What are the 3 basic mechanisms for regulating transcription factor functions?
1.) Binding of small effector mlc
Ex: steroid hormones
2.) Protein-protein interactions
Ex: Formation of heterodimers & homodimers
3.) Covalent modifications
Ex: Phosphorylation of activators
Chromatin remodeling
Refers to dynamic changes in structure of chromatin that occurs during life cycle of a cell
ATP used to drive change in locations &/or compositions of nucleosomes in order to make DNA more or less transcriptable (can activate or repress)
What kind of changes are involved in chromatin remodeling?
Range from local alterations in positioning of 1 or a few nucleosomes to larger changes that affect chromatin structure over a longer distance
What do changes in nucleosome position and histone composition have to do with gene regulation?
Turning genes "on" or "off" also involves changing chromatin structure in order to affect ability of transcription factors to gain access to & bind their target sequences in promoter region
Chromatin can be in...
1.) Closed conformation--little to no tran
Chromatin remodeling complexes
ATP-dependent
Set of diverse multiprotein machines that reposition/restructure nucleosomes (carry out chromatin remodeling)
-->Must recognize nucleosomes & use ATP to alter their configuration
What has been shown about nucleosomes in cells that express a particular gene compared with cells in which the gene is inactive?
Nucleosomes change position
Ex: Fibroblasts do NOT express beta-globin gene, but reticulocytes DO express this gene due to the altering of nucleosome positioning in the promoter region (-500 to +200)
What are the 3 possible effects of ATP-dependent chromatin remodeling?
1.) Change in nucleosome position
2.) Histone eviction
3.) Replacement w/variant histones
Change in nucleosome position
Changes relative positions of a few nucleosomes or changes relative spacing of nucleosomes over long stretch of DNA
Histone eviction
Removes histone octamers--> creating gaps where nucleosomes are not found
Replacement w/variant histones
Changes composition of nucleosome by removing standard histones & replacing w/histone variants
What usually happens w/regard to histones?
Usually standard histones are incorporated into nucleosomes when new DNA is synthesized during S phase
Later, some standard histones are replaced by histone variants via chromatin remodeling complexes--> creating functionally specialized regions of chroma
Histone variants
Mutated histones
What is the key role of histone variants?
Regulate structure of chromatin, thereby regulating gene transcription
What else do histone variants do?
Functions in binding of kinetochore proteins at chromo centromere; required for proper segregation of euk chromos; found at specialized sites in certain cells; play a role in DNA repair
Core histones' structure
Consists of globular domain and flexible, charged amino-terminal tail
DNA wraps around globular domains; amino-terminal tails protrude from chromatin
What kind of changes can standard & variant histones undergo?
Particular amino acids in amino-terminal tails are subject to covalent modifications, including: acetylation, methylation, and phosphorylation
How do histone modifications affect level of transcription?
1 method is to directly influence the interactions btwn nucleosomes
Ex: Acetylation of core histones
Histone acetylation
Histone acetyltransferase attaches acetyl group (COCH3) to core histones--> eliminates (+) charge on lysine side chain--> disrupting electrostatic attraction btwn histone protein & (-) charged DNA backbone--> DNA less tightly bound to histones--> attracts
Histone code hypothesis
**Note the histone modifications occur in patterns recognized by proteins
The pattern of covalent modifications to amino-terminal tails (like a lang/code to specify chromatin alterations) provides binding sites for proteins, which will affect degree of tr
Do covalent modification patterns inhibit or stimulate transcription?
Depends...
1 pattern may attract proteins that inhibit transcription--> silencing transcription of genes in that region
A diff pattern of histone modifications may attract proteins such as chromatin-remodeling enzymes--> altering positions of nucleosomes
For activated genes or genes that can be activated, where is the core promoter located?
NFR, which is about 150 bp in length
Nucleosome Free Regions (NFRs)
Euk have NFRs at beginning & end of genes
Needed for transcription to begin, but not sufficient to activate genes by itself; also important in transcription termination
At beginning of gene--NFR is region btwn -1 & +1 nuclesomes (usually containing varian
What are 3 things transcriptional activation involves?
Changes in nuclesome...
1.) Locations
2.) Compositions
3.) Histone modifications
What is the key role of many transcriptional activators?
Recruiting chromatin-remodeling enzymes & histone-modifying enzymes to promoter region
Describe transcriptional activation
see figure 15.13
DNA methylation
Modification of DNA structure in which methyl groups (CH3) are covalently attached to DNA
Usually inhibits transcription in euks, esp in case of methylating CpG sites
DNA methylation usually occurs on the ______? What's its effect on transcription?
Cytosine base
Usually inhibits transcription
DNA methyltransferase
Attaches methyl group (CH3) to 5 position of cytosine base--> forming 5-methylcytosine
CpG islands
Occur near many promoters of genes
When methylated--> blocks activator from binding to enhancer near the promoter, thus inhibiting transcription
When unmethylated--> transcription occurs
Refers to dinucleotide of C & G (connected by phosphodiester linkage
Housekeeping genes
Encode proteins needed by most cells in multicellular organism, thus expressed in most cell types--> meaning unmethylated CpG islands
Tissue-specific genes
Highly regulated & expressed only in particular cell types--> meaning methylated CpG islands
What role does DNA methylation play in cell differentiation?
All cells have same DNA, & so should be same, but we have multicellular species b/c of gene regulation which allows for genes to be turned "on" or "off"--the diff patterns of which genes are expressed & which are not facilitates cell differentiation, henc
How does DNA methylation inhibit transcription?
1.) Methylation of CpG islands near promoters may prevent or enhance (usually the first) binding of regulatory proteins; methyl group protrudes into major groove of DNA, preventing binding of activator
-->Methylated regions downstream from core promoter d
What does histone deacetylase have to do with?
Removes acetyl groups--> making it difficult for nucleosomes to be evicted from DNA, thus DNA is in closed conformation (more compact)--> transcription inhibited
What are the ways in which gene expression is commonly regulated after RNA is made?
These are the diff effects that take place to regulate gene expression via post-transcription RNA processing & translation...
1.) Alternative splicing
2.) RNA stability
3.) RNA interference
4.) General regulation of translation
5.) Translation regulation
Alternative splicing
Certain pre-MRNAs can be spliced in diff ways--> prod diff amino acid sequences means possibly prod diff polypeptides w/diff functions (thus 1 gene can prod multiple polypeptides, so organism can carry fewer genes in its genome--nature loves efficiency!!)
Constitutive exons
Always found in mature mRNA from all cell types
Needed for the general structure & function of the protein
Alternative exons
Not always found in mature mRNA (after splicing has occurred)
May subtly change structure, & therefore function of the protein in order to meet needs of particular cell type
Splicing factors
Splicing repressor--binds to 3'- splice site of an exon & prevents spliceosome from recognizing site; spliceosome moves on to next available 3'- splice site; result = exon is skipped (not spliced) & thus not included in mRNA
Splicing activator--binds to 3
RNA stability
Stability of mRNA influences mRNA concentration, thereby affecting gene expression (greater concentration = greater expression; lesser concentration = vice versa)
Stability may be regulated such that mRNA half-life is shortened or lengthened; a couple of
PolyA tail
Recognized by polyA-binding protein--enhances RNA stability
Over time, polyA tail shortens due to cellular exonucleases--eventually polyA tail is too short for polyA-binding protein to bind--> mRNA rapidly degrades by exo- & endonucleases
So, longer polyA
Destabilizing elements
Certain mRNAs (esp w/short half-lives) contain sequences acting as destabilizing elements commonly located in 3'-UTR (UnTranslated Region)--near 3' end, btwn stop codon & polyA tail
Ex: AU-rich element (ARE)--found in many short-lived mRNAs; consensus seq
RNA interference
Double stranded RNA can silence expression of mRNA--don't need to know details
Mediated by microRNA (miRNA) or short-interfering RNA (siRNA) via RNA-induced silencing complex
miRNA
Partially complementary to certain cellular mRNAs & inhibits their translation
miRNA is not coded by a structural gene, meaning it is not translated into a protein
siRNA
Usually perfect match to specific mRNAs & causes mRNAs to be degraded
Also not coded by structural gene, thus not translated into protein
How do miRNAs & siRNAs cause silencing of specific mRNAs?
Steps...
1.) Pre-miRNA or pre-siRNA is tanscribed from a gene; forms hairpin structure (the pre-mi/siRNA is looped such that it is double-stranded--see figure 15.24)
2.) Dicer (an endocnuclease) cuts hairpin structure--yielding double-stranded RNA w/out a
RNA-induced silencing complex (RISC)
Complementarity of mi/siRNA to specific regions of mRNAs allows RISC to recognize & bind to those mRNAs; 2 diff things may result...
1.) If mi- & siRNA are perfect match or high complementarity--> RISC directs degradation of mRNA
2.) miRNA is not perfect
3 benefits of RNA interference (RNAi)
1.) Defense mechanism against viruses
2.) Defense mechanism against possibly harmful transposable elements
3.) Normal cellular process for regulating gene expression
Phosphorylation
Mechanism of translational gene regulation
Translation initiation factors (IFs) are needed to begin protein synthesis; eIF2 alpha subunit & eIF4F play central role in controlling initiation of translation--phosphorylation of these factors have opposite ef
Phosphorylation of eIF2a
Inhibits translation
Activation of eIF2a protein kinases phosphorylates eIF2a--> causing eIF2a to bind tightly to another initiation factor subunit eIF2B--> preventing eIF2B from playing its role in binding initiator tRNA^Met to 40S ribosomal subunit--> i
Phosphorylation of eIF4F
Stimulates translation
Modulates binding of mRNA to ribosomal initiation complex--> promoting translation
DO WE NEED TO KNOW ABOUT THE CONDS IN WHICH THESE DIFF IFs ARE PHOSPHORYLATED??
Regulation of specific mRNAs
Involves RNA-binding proteins that directly affect translational initiation or RNA stability
Ex: Regulation of iron assimilation
Importance of iron
Essential element for organisms--required for function of several diff enzymes
How mammalian cells take up iron
Fe3+ in bloodstream binds to transferrin (protein that carries Fe3+ throughout bloodstream); transferrin-Fe3+ complex is recognized by transferrin receptor on cell's surface & bound to it; complex transported into cytosol by endocytosis; once inside, Fe3+
Regulation of iron assimilation in mammals
Involves RNA-binding protein called--iron regulatory protein (IRP)
IRP binds to iron response element (IRE)
IREs in mRNAs that encode ferritin & transferrin receptor are located in diff areas...
--> Binding of IRP to IRE of ferritin mRNA inhibits translat
Regulation of ferritin mRNA when iron concentration is low
IRP binds to IRE in 5'-UTR & inhibits translation
Regulation of ferritin mRNA when iron concentration is high
IRP binds to iron & is released from IRE; translation occurs
Regulation of transferrin receptor mRNA when iron concentration is low
IRP binds to IRE in 3'-UTR & enhances stability of mRNA (translation occurs)
Regulation of transferrin receptor mRNA when iron concentration is high
IRP binds to iron & is released from IRE; mRNA is degraded by endonucleases (translation inhibited)
Mutation
Heritable change in genetic material (structure of DNA permanently changed)
Chromosome mutations
Changes in chromo structure & #
Genome mutations
??????Permanent change in DNA sequence????
Single-gene mutations
Relatively small change in DNA structure that affects single gene
Point mutations
Change in single-base pair w/in DNA
Ex: base substitutions--transitions & transversions
Base substitutions
One base is substituted for another
Transitions
Pyrimidine changed to another pyrimidine (i.e. C to T) or purine changed to another purine (i.e. A to G)
More common than transversions
Pyrimidines = C & T
Purines = A & G
Transversions
Purines & pyrimidines interchanged (i.e. T to G)
What are the diff point mutations that could occur w/in coding sequence of a structural gene?
1.) Silent mutation
2.) Missense mutation
3.) Nonsense mutation
4.) Frameshift mutation
Silent mutations
Do NOT alter amino acid sequence of polypeptide--> no effect on protein function
Results from base substitution in wobble position (3rd base of codon)--degenerate DNA code means that if nucleotide sequence changes, the amino acid sequence may still be the
Missense mutations
Base substitution (more likely to be the 1st or 2nd base of codon) in which 1 amino acid is changed--> either neutral or inhibitory effect on protein function
**If no detectable effect on protein = neutral mutation
Ex: Sickle-cell anemia--mutated beta-glo
Nonsense mutations
Base substitution in which normal codon is changed to stop codon, altering many amino acids (how many depends on how early or late in the sequence the mutation occurs)--> prod truncated polypeptide due to early termination of translation--> inhibitory eff
Frameshift mutations
Addition or deletion of a # of nucleotides not divisible by 3--> shifts reading frame--> completely diff amino acid sequence downstream from mutation (# of altered amino acids also depends on how early the mutation occurs)--> inhibitory effect on protein
Neutral mutations
Result in no detectable effect on protein function; may include missense mutations & silent mutations
What are the consequences of a mutation that occurs in a non-coding region? What are those regions?
Promoter--> may incr & decr transcription rate
Regulatory element/operator site--> may disrupt ability of gene to be properly regulated
5'-UTR/3'-UTR--> may alter ability of mRNA to be translated; may alter mRNA stability
Splice recognition sequence--> ma
Up promoter mutation
Alteration of core promoter sequence that makes it more like the consensus sequence--> incr transcription rate
Down promoter mutation
Alteration of core promoter sequence that causes it to look less like consensus sequence--> decr transcription rate
Mutation in regulatory element, like an operator, does what?
Ex: Mutated lacO^c--prevents binding of lac repressor protein--> lac operon constitutively expressed (always on)--selective disadvantage b/c energy wasted expressing proteins not needed
Mutation in untranslated regions of mRNA (5'-UTR & 3'-UTR) may do what?
May affect phenotype by affecting gene expression via altering ability of untranslated region to be translatable or by altering its stability
Mutation in splice recognition sequence may do what?
In euk genes
Alters splice junctions & affects order or # of exons contained w/in mRNA
Wild-type
Relatively prevalent genotype or phenotype w/in natural population
Mutant allele
Rare mutation in which wild-type genotype is changed by altering DNA sequence of a gene (exactly what is sounds like--a mutated allele!)
**Categorizing mutation based on genotypic effect--ex: reversions
Reversions
Reverse mutation in which mutant allele is changed back to wild-type allele
Deleterious mutations
Decr chances of survival & reprod; more common
**Categorizing mutation based on phenotypic effect--ex: lethal mutations
Lethal mutations
Results in death of cell or organism (extreme type of deleterious mutation)
Beneficial mutations
Incr chances of survival & reprod; less common
**Categorizing mutation based on phenotypic effect
**Genotype &/or environmental conds may determine whether or not an allele is deleterious or beneficial (ex--sickle cell allele)
Conditional mutations
Affect phenotype only under defined set of conds
**Categorizing mutation based on phenotypic effect--ex: temperature-sensitive (ts) mutations
Suppressor mutations (AKA just: suppressors)
Second site mutation that affects phenotypic expression of 1st mutation--as the name suggests, it suppresses the affects of the 1st mutation (like a reversion, only at diff site from 1st mutation)
Classified according to relative location w/regard to 1st
Intragenic suppressors
Second mutant site is w/in the same gene as the 1st mutation
Involves change in protein structure that compensates for abnormality in protein structure caused by 1st mutation
Ex: Lactose transport function
Intergenic suppressors
Second mutant site is w/in diff gene than the one where the 1st mutation occurred
These suppressors work in a variety of ways, involving...
1.) Redundant function
2.) Common pathway
3.) Multimeric protein
4.) Transcription factor
Redundant function (intergenic suppressors)
1st mutation inhibits function of protein; 2nd mutation alters diff protein to carry out that function
Common pathway (intergenic suppressors)
2 or more diff proteins may be involved in a common pathway; 1st mutation causes defect in 1 protein may be compensated for by 2nd mutation that alters function of a diff protein in same pathway
Multimeric protein (intergenic suppressors)
1st mutation in gene encoding 1 protein subunit that inhibits function may be suppressed by 2nd mutation in diff gene that encodes a diff subunit of the protein--restoring function
Transcription factor (intergenic suppressors)
1st mutation causes loss-of-function of a particular protein; 2nd mutation may alter transcription factor, causing it to activate expression of another gene--this other gene encodes a protein that can compensate for loss-of-function caused by 1st mutation
How might changes in chromosome structure affect expression of a gene?
Change in chromo structure is associated w/an alteration in expression of SPECIFIC genes
-->Breakpoint
-->Position effect due to inversions & translocations
Breakpoint
Region where 2 chromo pieces break & rejoin w/other chromo pieces
When breakpt occurs w/in middle of a gene--> likely to inhibit gene function b/c separates 1 gene into 2 pieces
Position effect
Result of gene moving from 1 location to another (but gene is still intact)
Inversions
Position effect due to regulatory sequences...
A gene is moved next to a regulatory sequence (i.e. silencers or enhancers) for a diff gene, that influence expression of relocated gene (all of this occurs on the same chromo
Translocations
Position effect due to translocation to a heterochromatic chromo...
A gene from a euchromatic (less condensed) chromo is moved to a heterochromatic (highly condensed) chromo--> thereby "turning off" expression of gene now in condensed region
May prod vari
Germ-line mutations
Germ line = cells that give rise to gametes
Mutation can occur directly in egg or sperm cell, or occur in precursor cell that prod gametes
If mutant gamete participates in fertilization--> all cells of offspring will contain mutation
Somatic mutations
Somatic cells = all the cells excluding germ-line cells
Mutation can occur in early or late stages of dev...
-->Size of affected region depends on timing of mutation--usually earlier the mutation occurs during dev, then the larger the affected region
Genetic mosaics
Indiv that has somatic regions that are genotypically (& consequently sometimes phenotypically) diff from each other
Spontaneous mutations
Changes in DNA structure caused by abnormalities in biological processes
May result in chromo mutations, genome mutations, & single-gene mutations
Common causes...
Aberrant recombination
Aberrant segregation
Errors in DNA replication
Transposable elements
Induced mutations
Changes in DNA structure caused by environmental agents (called mutagens)
Common causes...
Chemical agents
Physical agents
Luria & Delbruck experiment
Wanted to determine if mutations occur randomly/spontaneously or as a result of environmental conds: physiological adaptation hypothesis (acquired inherited resistance hypothesis) vs. spontaneous mutation hypothesis
Experimental system involved resistance
Physiological adaptation hypothesis (acquired inherited resistance hypothesis)
If true, then rate of adaptation to phage should be constant, depending upon exposure to the bacteria
Spontaneous mutation hypothesis
If true, rate of adaptation should vary widely, depending upon when random mutation occurred in population
What did Luria & Delbruck observe?
Random plates (containing T1 phage) were able to grow bacteria (b/c mutation occured that made bacteria resistant to phage) & how much bacteria grew varied--some had no bacteria, others a little, and some grew lots--> observations support spontaneous muta
Mutation rate
Likelihood that a gene will be altered by a new mutation; is expressed as the number of new mutations in a given gene per generation
Mutation frequency
# of mutant genes divided by the total # of genes w/in a given population
Depurination
Involves removal of purine (A or G) from DNA
How it happens--covalent bond btwn deoxyribose & purine base is somewhat unstable & occassionally undergoes spontaneous rxn w/water that releases base from sugar--> creating apurinic site
Replication at apurinic site
Since there is no complementary base present to specify incoming base for new strand, any of the 4 bases are added to new strand in region opposite of apurinic site (may lead to mutation)
What happens in future rounds of replication involving apurinic site?
...
Deamination of cytosine
Involves removal of amino group from cytosine base, (other bases not readily deaminated) converting it to uracil (which should not be in DNA!!)
Mutation may result b/c uracil will then bond w/adenine
Originally...
Template strand had C--> so daughter stra
Deamination of 5-methylcytosine
Involves removal of amino group from methylated cytosine base, converting it to thymine
Mutation hot spots--more likely to cause mutation than deamination of unmethylated cytosines, b/c thymine is natural constituent of DNA & so DNA repair mechanisms cann
What happens in future rounds of DNA replication involving deaminated cytosines or deaminated 5-methylcytosines?
...
Tautomeric shifts
Temporary change in base structure by which mutation may arise
Tautomers = bases which exist in keto & enol, or amino & imino forms
-->Common, stable form of G & T = keto
---->Relatively small amts of G & T exist in enol form
-->Common, stable form of A &
What happens in future rounds of replication involving a tautomeric shift?
...
Reactive Oxygen Species (ROS)
Prods of oxygen metabolism in all aerobic organisms
When accumulated--> damage cellular molecules including: DNA, proteins, & lipids
Various enzymes prod by cells & certain chems obtained in diet act as antioxidants to combat/breakdown ROS
Oxidative stress
Refers to imbalance btwn prod of ROS & an organisms ability to break them down
ROS overaccumulate can cause oxidative DNA damage
Oxidative DNA damage
Changes in DNA structure caused by ROS (can be spontaneous or via environmental agents--UV light, X-rays, harsh chems)
Guanine bases are particularly vulnerable to oxidation--> 1 possible prod is 8-oxoguanine (8-oxoG), which pairs w/A instead of C!!
Origi
What is the result of the mismatch involved in guanine oxidation?
GC base pair becomes TA base pair (transversion mutation)
What are a couple of ways in which mutagens alter the structure of DNA?
Some mutagens covalently modify DNA structure
-->Ex: Nitrous acid
Some mutagens are incorporated into DNA strands during replication
-->Ex: 5-Bromouracil
Nitrous acid (HNO2)
Chemical mutagen
Deamination occurs--HNO2 replaces amino groups (NH2) w/ keto groups (O)
-->Changes C to U
-->Changes A to hypoxanthine
Altered DNA replicates, modified bases do not pair w/appropriate bases...
-->U w/A
-->hypoxanthine w/C
CONFUSED B/C U &
What happens in future rounds of DNA replication involving exposure to HNO2?
...
5-Bromouracil (5-BU)
Chemical mutagen
Thymine analog that base-pairs w/adenine, but due to tautomeric shifts--5BU may base-pair w/guanine (this occurs at relatively high rates; transition mutation)
Originally...
Template strand had 5-BU--> so daughter strand would have A
Afte
What happens in future rounds of DNA replication involving 5-Bromouracil exposure?
...
Ionizing radiation
Physical mutagen
Creates free radicals that can cause base deletions, single nicks in DNA strands, cross-linking, & chromosomal breaks
Nonioninzing radiation
Physical mutagen
Causes formation of thymine dimers--creating problems during replication
Ex: UV light, like UV-B exposure can lead to thymine dimers that cause melanoma
Why are DNA repair systems important?
Vital to survival of organisms b/c most mutations are deleterious--if even 1 repair system is missing, could cause phenotypic effect
DNA repair systems
Protein based; involve several steps...
?????
Direct repair
Enzyme recognizes incorrect alteration in DNA structure & directly converts it back to correct structure
Direct repair of thymine dimers
Mutation to correct--UV light causes formation of thymine dimers
Photolyase recognizes thymine dimers & splits them, returning DNA to original structure
Direct repair of methylated bases
Mutation to correct--G bases alkylated (methylated/ethylated) by agents such as nitrogen mustard & EMS
Alkyltransferase removes methyl or ethyl groups from G bases & transfers them to cysteine side chain w/in the alkyltransferase protein (means each alkly
Base excision repair (BER)
Steps...
1.) N-glycosylase recognizes abnormal base & cleaves bond btwn base & sugar--leaving behind an apurinic or apyrimidic site
2.) AP endonuclease recognizes missing base & cleaves/nicks DNA backbone on 5' side of missing base
3.) 3 possibilities dep
DNA N-glycosylase
Organism prod diff types in order to recognize specific abnormalities w/in DNA structure such as: uracil, 3-methyladenine, 7-methylguanine, & pyrimidine dimers
Describe the 3rd step in BER if the species is bacteria, like E. coli
Process call nick translation occurs (even though its replication, not translation)--DNA polymerase I uses it 5' to 3' exonuclease activity to remove damaged region; at same time it replaces the region w/normal nucleotides
Describe the 2 different possible 3rd step in BER if the species is eukaryotic, like humans
1.) DNA polymerase beta removes apyrimidic or apurinic nucleotide & replaces it w/correct nucleotide
2.) DNA polymerase delta or epsilon can synthesize short segment of DNA--> generating a flap; flap is removed by endonuclease
Nucleotide excision repair (NER)
Can repair various types of DNA damage, including: thymine dimers, chem modified bases, missing bases, & certain types of crosslinks
Found in all euks & proks
Damaged strand removed from DNA; intact strand used as template for resynthesis of normal comple
How does NER work in prokaryotes, like E. coli, in repairing thymine dimers?
Steps...
1.) UvrA-UvrB complex tracks along DNA in search of damaged DNA (distorted double helix)
2.) After damage is detected-->UvrA is released & UvrC binds to the site
3.) UvrC makes cuts on both sides of thymine dimer
4.) UvrD (a helicase) removes dam
What are some diseases that result from lack of NER?
Xeroderma pigmentosum; Cockayne syndrome; PIBIDS syndrome
BER vs. NER
BER--removes damaged BASE
NER--removes damaged DNA SEGMENT
Mismatch repair system
Recognizes & corrects base pair mismatch
Exists in ALL species
Net result = mismatch corrected by removing incorrect region in daughter strand; resynthesizes the correct sequence using parental DNA as template
How does the mismatch repair system work in bacteria, like E. coli?
Steps...
1.) Protein MutS locates mismatch
2.) MutS forms protein complex w/MutL; MutL acts as linker that binds to MutH, which is bound to a hemimethylated site; due to distance btwn MutS & MutH, a loop is formed whilst linked--> stimulating MutH
3.) Mut
How does a DNA repair system determine which base to remove if mismatch is due to an error in DNA replication?
Prior to DNA, parental DNA has been fully methylated; immediately after DNA replication, new DNA is only hemimethylated--providing a way for the repair system to distinguish btwn parental strand & daughter strand (which contains the incorrect base)
Transcription-coupled DNA repair
Phenomenon in which actively transcribed DNA is more efficiently repaired (repaired at faster rate) than non-transcribed DNA--may be due to less compact structure of transcribed DNA
How are actively transcribed regions targeted by DNA repair systems?
Protein in E. coli called transcription-repair coupling factor (TRCF) targets NER system to actively transcribe genes w/damaged DNA
How does transcription-coupled DNA repair work to fix a thymine dimer in bacteria, like E. coli?
Steps...
1.) RNA polymerase is transcribing along its track, until it encounters a thymine dimer--> stalls RNA polymerase
2.) TRCF (a helicase) removes RNA polymerase from damaged template strand
3.) TRCF has binding site for UvrA--allowing for recruitmen
Recombinant DNA molecules
Covalently linked DNA fragments from 2 diff sources
What has recombinant DNA technology enabled researchers to study? What are some applications of this technology?
Use of in vitro mlcular techniques allows scientists to manipulate DNA fragments to prod new arrangements
Figure out relationships btwn gene sequences & phenotypic consequences
Gene therapy, screening for human diseases, recombinant vaccines, & prod of tr
Why do molecular biologists frequently focus on the structure & function of proteins, or the genes that encode them?
B/c proteins are workhorses of cells & b/c they are prod of genes
Gene cloning
Making many copies of a gene
Uses of gene cloning
1.) Gene sequencing--gene cloning provides enough DNA to sequence the gene--revealing promoter, regulatory sequences, & coding sequences; also helps ID cancer & other inherited diseases
2.) Site-directed mutagenesis--purposefully mutated gene in order to
Chromosomal DNA
ONE type of DNA that is needed in cloning experiment--it is the DNA that contains the gene of particular interest (the gene that you want to clone)
Vector DNA
DNA resulting from removal of DNA segment from native site w/in a chromo & inserted into a smaller segment of DNA known as a vector (small piece of DNA mlc that replicates independently of host cell's chromosomal DNA & prod many identical copies of insert
Host cell
Cell harboring a vector
Plasmids
Most vectors are derived from plasmids
Bacteria & some euks contain plasmids--small circular pieces of DNA
Commercially available plasmids have unique sites where scientists can easily insert DNA fragments
R factor plasmids
Plasmid that carries gene that resists antibiotics or other toxic substances
Plasmid's origin of replication
DNA replication is like that in bacteria b/c plasmid is circular & contains only 1 origin of replication
This sequence is recognized by restriction enzymes of host cell & allows replication to occur--determining which cell types a vector can replicate in,
Selectable markers
Often in vectors, they are resistance genes (often found in cloning vectors) that provide host cell w/ability to grow in presence of toxic substance
Viral vector
Chromosomal gene inserted into viral genome-->that way gene of interest is replicated whenever viral DNA is replicated
Cosmids, BACs, & YACs
Vectors used to clone large pieces of DNA
Restriction enzymes/endonucleases
Cuts DNA
Used in cloning experiments
Specific restriction enzyme binds to specific base sequence; cleaves DNA backbone in 2 places (1 in each strand)
Digest DNA into fragments w/"sticky ends"
Usually recognize palindromic sequences
Palindromic sequence
Sequence in 1 strand that is identical to the complementary strand, when read in opposite direction
Ex: 5'-GAATTC-3' & complementary strand: 3'-CTTAAG-5' & when read in opposite direction: 5'-GAATTC-3'
Sticky ends & their purpose in recombinant DNA technology?
DNA fragments made by digestion of DNA by certain restriction enzymes
Single-stranded regions of DNA that can H-bond to complementary sequence of DNA from a diff source--this interaction is temporary, need...
Role of ligase in recombinant DNA technology?
Makes interaction of "sticky ends" & complementary sequence permanent by catalyzing the covalent linking of the sugar-phosphate backbones of the 2 DNA strands
Gene cloning process
1.) Insertion of DNA fragments into vectors
2.) Propogation w/in host cells
Inserting DNA fragment into vector
a.) Restriction enzymes cut out gene of interest from chromosomal DNA & cuts up the rest of DNA into many fragments; the same restriction enzymes recognize & cut the UNIQUE (only 1) restriction site in plasmid's DNA
b.) Digested (cut up) chromosomal DNA &
Propagation w/in host cell
a.) Recombinant DNA is mixed w/bacterial cells (that do not contain gene for resistance to ampicillin--no plasmid) that have been treated w/agent that makes them permeable to DNA (called competent cells)--the process of extracellular DNA being taken up by
Recombinant vector isolation--how do you distinguish btwn bacterial colonies containing a recircularized vector versus a recombinant vector?
For ex given in Figure 18.2 of the textbook...
Chromosomal DNA was inserted into lacZ gene, which encodes beta-galactosidase enzyme--> recombinant vector disrupts lacZ gene, preventing it from prod functional enzyme; recircularized vector contains intact
What do you do after isolating bacterial colonies containing recombinant vector?
You determine which bacterial colony contains recombinant vector w/the gene of interest!!! DO WE NEED TO KNOW HOW THIS IS DONE, B/C THE BOOK DOESN"T EXPLAIN IT, NOR DOES PPT??
cDNA (complementary DNA)
DNA made from RNA as a starting material
How is cDNA made?
Made from mRNA via reverse transcriptase...
1.) mRNA is purified from sample of cells & then mixed poly-dT primer
2.) Reverse transcriptase & deoxyribonucleotides (dNTPs) are added to make complementary DNA strand to that of the mRNA
3.) Add RNaseH to cut
Poly-dT primer
Oligonucleotide (fancy name for a short strand of DNA) that contains primer sequence made up of a string of thymine-containing nucleotides
It is complementary to mRNA's polyA tail, allowing the primer to bind to mRNA's 3' end--this is the starting point f
RNaseH
Partially digests RNA in a way that generates short RNA strands that are used as primers by DNA polymerase (they are called RNA primers, even though DNA uses them)
Why is cDNA cloning useful?
cDNA lacks introns, making it simpler to insert cDNA into vectors for cloning, which is nice from research perspective--esp those trying to determine the coding sequence of a particular structural gene
Polymerase Chain Reaction (PCR)
Method of gene cloning w/out aid of vectors & host cells
Used to make a large amt of DNA in a defined region (gene of interest area) that is flanked by 2 primers
How does PCR work?
1.) Primers bind to specific site in DNA b/c their bases are complementary at those sites--1 primer at each end of the gene of interest in the chromosomal DNA
2.) End result of PCR = amplification of gene of interest (region flanked by primers)
-->Amplifi
How do you get primers w/sequences that flank the gene of interest?
Primers are key reagents in PCR & in order to ensure that you use the right primer you must already know something about the nucleotide sequence of the gene of interest
Researchers look up sequence of region containing gene of interest & decide where they
How do you know the sequence of the gene of interest?
B/c entire genome of many species has already been determined--this sequence info can be found in genome database of the species of interest
In addition to primers & DNA containing gene of interest (which is called template DNA), what else does PCR require?
Deoxyribonucleoside triphosphates (dNTPs) & a thermostable form of DNA polymerase (i.e. Taq polymerase), which is necessary b/c PCR involves heating steps that would inactivate most other natural forms of DNA polymerase
-->Taq polymerase is isolated from
What are the 3 steps of PCR cycle? Why is this process called a chain rxn?
1.) Denaturation
2.) Primer annealing
3.) Primer extension
Process is repeated for many cycles, thus doubling amt of template DNA many times in a row
Called chain rxn b/c prods of each previous rxn (newly made DNA strands) are used as reactants (template
Denaturation
Separates DNA strands w/high temp
Primer annealing
Lower temp, allows primers to bind to template DNA
Primer extension
Incubate at slightly higher temp, which allows DNA synthesis to occur
What is PCR conducted in & what is its purpose?
A thermocycler--provides precise timing & temp controls
Typical PCR run usually involves 20 to 30 cycles
-->Assuming 100% efficiency--region btwn primers would incr 2^20-fold after 20 cycles (about a million fold) [doubles each cycle, so 2^n, where n = #
Reverse transcriptase PCR
Used to detect & quantitate amt of specific RNAs in a living cell
Extremely sensitive--can detect even small amts of RNA from a single cell
How does reverse transcriptase PCR work?
RNA is isolated from sample & mixed w/deoxyribonucleotides, reverse trasncriptase, & a primer that binds near 3' end of the RNA of interest
Result = generation of single-stranded cDNA, which can then be used as template DNA in conventional PCR rxn
What do molecular geneticists need to be able to identify?
Chromosome DNA has thousands of diff genes, so they need to be able to...
Specifically ID a gene w/in a mixture of many other genes or DNA fragments &...
ID gene prods, like RNA transcribed from a particular gene, or protein prod encoded by an mRNA
DNA library
Collection of DNA hybrid vectors that can be constructed using genomic DNA or cDNA
Genomic library
DNA library constructed from chromosomal DNA as starting material
mixed w/plasmid vectors or viral vectors
Result = collection of many colonies; each colony contains millions of cells derived from a single transformed cell (the transformed cells contain a
cDNA library
DNA library constructed from cDNA as starting material
cDNA that is made by reverse transcriptase (as already described) is inserted into vectors...
1.) Oligonucleotide linkers are attached to ends of cDNAs via ligase
-->Linkers contain DNA sequences w/un
How might a cDNA library be useful?
Since cDNA lacks introns, a researcher may use cDNA library to express encoded protein of interest in a cell that would not splice out introns properly
What is represented in the library?
Lots of diff bacterial colonies (each contains a diff hybrid vector), so only small percentage actually contains gene of interest (unlike PCR which copies the specified gene of interest only)
Scientists must have method to determine which colony has gene
Colony hybridization
Method for IDing bacterial colony that contains gene of interest by using a DNA probe
DNA probe
Radiolabeled or fluorescently labeled oligonucleotide, or fragment of DNA from cloned gene, or specific RNA
Use to detect recombinant vector w/gene of interest (probes must be specific--i.e. hybridizing w/a particular enzyme)
DNA w/in bacteria colony that
How do you obtain the specific probe needed?
Several ways--depends on whether the gene of interest has already been cloned...
1.) If gene has been cloned, then you can use a piece of the cloned gene as a radiolabeled probe
2.) If gene has not been cloned...
-->Use sequence similar to gene of interes
Southern blotting
Used to detect presence of specific gene in a mixture of chromosomal DNA fragments
Prior to Southern blotting, the gene of interest is cloned; cloned DNA labeled in vitro
Labeled DNA used as probe to detect presence of the gene or a homologous gene w/in m
Steps in Southern blotting
1.) Chromosomal DNA is digested w/a restriction enzyme
2.) Fragments separated by gel electrophoresis according to mlcular mass & then denatured by NaOH
3.) Blotting--bands w/in gel are transferred (blotted) to nylon membrane via traditional method or ele
High stringency
Procedure is done at high temps &/or low salt concentrations such that the probe DNA & chromosomal DNA fragment must be very complementary (nearly perfect match) to hybridize
Reveals how unique (or not unique) the gene of interest it
Low stringency
Procedure is done at low temps &/or high salt concentrations such that the probe DNA & chromosomal DNA fragment must be similar (but doesn't have to be perfect match) complementarity to hybridize
Reveals members of the gene family (that the gene of intere
Northern blotting (AKA: reverse-Southern blotting)
Used to detect specific RNA among mixture of many RNA mlcs; also to study transcription of genes at mlcular lvl--can determine if specific gene is active in a particular cell type or at a particular stage of dev; can also reveal if pre-mRNA transcript is
Autoradiography of smooth muscle cells (lane 1), striated muscle cells (lane 2), & brain cells (lane 3)
A band is present in each lane--> indicating that the particular mRNA of interest is made in all 3 cell types
Density of the band tells you the relative amt being made--band in lane 3 is less dense than that in lane 1 or lane 2, meaning there is less of t
Western blotting
Used to detect proteins & determine if specific protein is being made in a certain cell type or at a particular stage of dev
Similar to Southern blotting
How does Western blotting work?
Proteins must be treated w/sodium dodecyl sulfate (SDS) to denature the proteins & coat them negative charges
Proteins are run out on SDS-PAGE gel
PAGE = polyacrylamide gel electrophoresis
Uses antibodies as probes: antibodies bind to sites on mlcs called
What does the autoradiograph of the red blood cells (lane 1), brain cells (lane 2), & intestinal cells (lane 3) indicate?
In this ex the primary antibody recognizes beta globin protein
Band only appears in lane 1--> indicating that beta globin protein is being synthesized in red blood cells, but not in brain or intestinal cells
How may researchers study the binding of proteins to specific sites on DNA?
Gel retardation assays & DNase I footprinting procedures
Gel retardation is also used to study protein-RNA interactions
Gel retardation assay (AKA: gel mobility shift assay)
Used to determine if a protein binds to a specific DNA fragment or RNA mlc...
-->Commonly used to detect interactions btwn mRNAs & RNA-binding proteins, & interactions btwn euk transcription factors & DNA regulatory elements
Protein-DNA binding
Binding of a protein to DNA slows its migration through the gel
Figure 18.16 shows that the band on the left (not protein bound DNA) has moved further than the band on the right (protein bound DNA) b/c protein bound DNA is heavier
If concentration of DNA
DNase I footprinting
Tries to ID regions of DNA that interact w/a DNA-binding protein
Involves mlcular interactions of DNA, DNA-binding protein, & agents that alter DNA structure
Bound proteins protect DNA from DNase I degradation
????
Dideoxy method of DNA sequencing
Process uses dideoxyribonucleotides, which lack 3'-OH group--> preventing synthesis of DNA strand during PCR rxn & causing chain termination
How does dideoxy DNA sequencing work?
1.) Many copies of DNA to be sequenced, along w/primers, dNTPs, fluorescently labeled ddNTPs, & DNA polymerase are all mixed together & incubated to allow synthesis of DNA
2.) Newly made strands are then separated by gel electrophoresis
Result = sequence
What is a faster/more practical way to deduce sequence from gel?
Automated procedure using laser & fluorescent detector
Site-directed mutagenesis
Analysis of mutations can provide useful info about genetic processes, gene expression, & phenotype
Allows researchers to introduce mutations into cloned genes or other DNA segments
Altered gene can be introduced into living organism to examine how mutati