Genetics exam #2

diphtheria facts

* sore throat, fever, coma and death
* epidemics in the 1920s killed 15,000 people in the US, mostly children
* Corynebacterium diphtheriae: toxin targets elongation factor 2 (EF2), stops translation
* we don’t have epidemics today due to vaccinations

proteins

* synthesized from 20 amino acids
* composed of 1 or more polypeptides
* one string of amino acids linked together by peptide bonds
* amino acid structure

nonpolar, , aliphatic R groups

* Glycine (Gly, G)
* Alanine (Ala, A)
* Valine (Val, V)
* Leucine (Leu, L)
* Isoleucine (Ile, I)
* Proline (Pro, P)

Polar, uncharged R groups

* Serine (Ser, S)
* Threonine (Thr, T)
* Cysteine (Cys, C)
* Methionine (Met, M)
* Asparagine (Asn, N)
* Glutamine (Gln, Q)

Aromatic R groups

* Phenylalanine (Phe, F)
* Tyrosine (Try, Y)
* Tryptophan (Trp, W)

Positively charged R groups

* Lysine (Lys, K)
* Arginine (Arg, R)
* Histidine (His, H)

Negatively charged R groups

* Aspartate (Asp, D)
* Glutamate (Glu, E)

types of protein structures

primary, secondary, tertiary, quaternary

peptide bond

covalent bond formed between 2 amino acids

Francis Crick and James Watson

described the double-helix structure of DNA

genetic code is composed of nucleotide triplets

these three nucleotides are a codon, which specifies one amino acid

genetic code is non overlapping

each nucleotide belongs to one codon, which are read consecutively

genetic code is degenerate

all but two AA are specififed by more than one codon, usually 3rd base differs

ex: Arginine, CGU, CGC, CGA, CGG

wobble

a base in the 1st position of an anticodon can pair with any of several bases in position of 3 of codon

first position of tRNA

5’ end

third position of mRNA

3’

tRNA to mRNA bases

5’ to 3’

* A = U
* C = G
* G = C or U
* U = A or G
* I = A, U, or C

genetic code is ordered

codons for amino acids with similar chemical properties are closely related to each other

genetic code has start and stop codons

* start codon: AUG
* stop codons: UAA, UGA, UAG

genetic code is (almost) universal

same in viruses, bacteria, and humans

Nirenberg and Matthaei and Leder and Khorana

* the first to link specific coding sequences to specific amino acids


* laid cornerstone for the complete analysis of genetic code
* synthesized polypeptides in a cell free (in vitro) system for synthesizing proteins
* found means of producing synthetic mRNA to serve as templates for polypeptide synthesis in the cell-free system

in-vitro translation

* synthesized polymers of RNA
* UUU-phenylalanine
* CCC-proline

in-vitro translation process

* uracil nucleotides w/ polynucleotide phosphorylase = Poly(U) homopolymer
* precipitate protein
* free amino acids separate from protein
* suction for further distillation

4 parts of translation in prokaryotes

* tRNA charging
* initiation
* elongation
* termination

tRNA charging

aminoacyl-tRNA synthetase attaches correct amino acids to tRNA molecules (two step reaction, requires ATP)

initiation of translation

* IF1: binds to 30S subunit and prevents aminoacyl tRNA from binding to the A site
* IF2: binds f-met-tRNA to 30S-mRNA complex and hydrolyzes GTP
* IF3: binds to 30S subunit, preventing it from associating with the 50S subunit

elongation of polypeptide

* EF-Tu: binds GTP, beings aminoacyl tRNA to the A site of the ribosome
* EF-Ts: generates active EF-Tu
* EF-G: stimulates translocation, GTP-dependent

termination of translation and release of polypeptide

* RF1: catalyzes release of the polypeptide chain from the tRNA and dissociation of the translocation complex; specific for UAA and UAG termination codons
* RF2: behaves like RF1, specific for UGA and UAA codons
* RF3: stimulates RF1 and RF2

translation components

* small subunit and large subunit in ribosome in GTP
* initiation factors
* elongation factors
* release factors
* anticodon and initiator tRNA
* many triplet codons

initiation of translation

1. initiation factors bind to small subunits and attract mRNA
2. initiation complex: tRNA binds to AUG codon of mRNA in P site, forming initiation complex, IF3 is released
3. large subunit binds t complex; IF1 and IF2 are released. subsequent aminoacyl tRNA is poised to enter the A site

initiation

* must assemble components (ribosome, mRNA, fmet-tRNA, initiation factors, GTP)
* initiation factor 3 binds small ribosomal subunit (prevents large subunit from binding prematurely, IF1 assists)
* small subunit of ribosome binds to Shine Dalgarno sequence on mRNA
* IF2 forms complex with GTP
* fMet tRNA attaches to start codon on mRNA
* IF3 comes off, allowing large subunit to bind
* GTP hydrolyzes, IF1 and IF2 leave
* large ribosomal subunit binds
* tRNA-fMet is in P site

three binding sites for ribosomal subunit

* E = exit
* P = peptidyl
* A = aminoacyl

initiation: eukaryotes

* similar, but no shone Dalgarno
* 5’ cap is recognized
* slightly different initiation factors
* Poly A tail loops back to interact with proteins on the 5’ cap

Elongation

* EF-Tu brings charged tRNA to A site
* requires GTP hydrolysis
* EF-Ts generate EF-Tu/GTP complex

if codon and anticodon match:

1. peptide bond formed by peptidyl transferase (ribozyme)
2. translocation


1. ribosome moves 1 codon further
2. movement requires GTP hydrolysis and EF-G
3. uncharged tRNA is removed from E site


1. 2nd tRNA with growing peptide chain is in the P site, a site is empty
4. new charged tRNA enters A site, process repeats itself

termination

* ribosome moves until it gets to a stop codon
* released factors bind stop codons in A site
* GTP hydrolysis occurs to break polypeptide away from complex
* everything falls apart
* termination codon enters A site, RF1 or RF2 stimulates hydrolysis of the polypeptide from peptidyl tRNA
* ribosomal subunits dissociate and mRNA is released, polypeptide folds into native 3-D conformation of protein, charged tRNA released

coupling of transcription and translation

only in prokaryotes (translation begins before transcription is complete)

polycistronic mRNA

* only in prokaryotes
* mRNA contains info for more than 1 gene
* each different gene has its own shine dalgarno and AUG
* often composed of genes for the same pathway

why must antibiotics exploit differences between prokaryotic and eukaryotic cells?

* prokaryotic cells have exploitable differences from eukaryotic cells
* antibiotics like penicillin inhibit bacterial cell wall production with minimal effects on animal cells
* antibiotics like tetracycline inhibit prokaryotic ribosomes but have less effect in eukaryotes

puromycin

* looks like 3’ end of aminocyl tRNA
* binds A site, peptidyl transferase creates a bond between peptide and puromycin
* no more elongation, peptide released

streptomycin

* binds to small ribosome subunits
* blocks initiation of translation

tetracycline

prevents aninoacyl tRNA from binding to A site

chloramphenicol

* binds large subunit
* blocks peptidyl transferase activity

gene regulation in prokaryotic cells

* no differentiation
* growth and survival

genes are regulated primarily by:

* transcriptional control
* RNA stability
* translational control
* posttranslational control (protein function)

inducible systems

synthesis of enzymes occurs only when substrate is present

repressible systems

synthesis of enzymes stops when product is not needed, detect excess of end products

inducible enzymes

made in response to a particular stimulus

constitutive enzymes

made continuously

lactose operon of E. coli

* sequence of adjacent genes under the transcriptional control of the same promoter and operator
* E.coli grows best using glucose as a carbon sources
* will grow on lactose

additional enzymes required to grow E.coli on lactose

* normally transcribed at a very low rate
* induced in the presence of allolactose

LacZ operon

* B-galactosidase
* splits lactose into glucose and galactose
* converts lactose into allolactose

lacY operon

* lactose permease
* active transport of lactose into cell

LacA operon

* transacetylase
* function not well understood

LacZ, Y, and A are what?

polycistronic (deciphers various proteins and is an attribute of many prokaryotic bacterial and chloroplast mRNAS, carried information for the synthesis of more than one protein)

Lac operon

* grow E.coli in glucose
* basal level of txn
* 3 molecules of B-gal per cell
* switch to lactose
* induced
* 3000 molecules of B-gal per cell
* Lac I
* produced constitutively
* produces laci repressor

the component of the wild-type lac operan and the response in the absence and presence of lactose

repressor gene, promoter, operator gene, leader, structural genes Z + Y + A

absence of lactose

* Lacl protein binds lac operator
* RNA pol can not bind promoter, cannot initiate txn
* Lac operon is off
* NOTE: low level of txn reflects the fact that repressor does not bind irreversibly

presence of lactose

* B-gal converts some lactose to allolactose (inducer)
* allolactose binds repressor (allosteric shift)
* repressor dissociates from DNA
* RNA pol now initiates txn

the lac operon in the absence of lactose

* lacl protein binds the lac operator
* if RNA polymerase cannot bind promoter, then transcription cannot be initiated
* this means the lac operon is OFF
* low level of transcription reflects the fact that repressor does not bind irreversibly

the lac operon in the presence of lactose

* B-galactose converts some lactose into allolactose (inducer)
* allolactose binds repressor (allosteric shift)
* repressor dissociates from DNA
* RNA polymerase now initiates transcription

mutations in lac operon system

* promoter of lac operon:
* so RNA pol cannot bind
* different results in glucose and lactose
* operator
* delete operator sequence
* Lacl
* delete gene for lac I
* or over-express lac I

catabolite repression

* if both glucose and lactose are present, glucose will be used

catabolite repression with glucose

* adenyl cyclase inhibited by glucose
* not as much as CAP-cAMP

catabolite reaction with no glucose

* cAMP made by adenyl cyclase
* CAP binds cAMP
* CAP-cAMP binds DNA at CAP site (upstream of lac promoter)
* DNA bends, RNA poly binds more strongly

catabolite repression with absent glucose

* glucose absent
* CAP (catabolite activating protein) + cAMP
* =
* promoter region (includes CAP-binding site and polymerase site)
* structural genes (transcription and translation occur)

cAMP facts

as cAMP levels increase, cAMP binds to CAP, causing an allosteric transition

catabolite repression with present glucose

* glucose
* CAP means cAMP levels decrease
* =
* CAP cannot bind efficiently
* RNA polymerase seldom binds
* transcription and translation diminished

tryptophan operon

* repressible system
* turned off when end product builds up
* 5 structural genes A-E, promoter, operator, 162 bp leader region

trpR

repressor located away from operon

components of repressible operon

* promoter, operator, leader, and attenuator
* repressor gene before regulatory region and structural genes in trp operon

repressor proteins have what?

tryptophan binding site

with tryptophan abundant:

* tryptophan binds repressor, repressor binds operator, txn reduced 70x

without tryptophan

* repressor inactive
* txn occurs (attenuation possible)

attenuation

* involves leader region between operator and 1st structural gene
* mRNA region termed leader transcript
* four subregions 1-4
* can form stem loops
* includes tryptophan codons

in the attenuation model, tryptophan operon is regulated by:

changing direction

attenuation with tryptophan

* txn, tln proceed up leader peptide gene
* ribosome overlaps region 2
* 3-4 terminator stems forms
* txn terminated

attenuation without tryptophan

* ribosome pauses at trp codon
* 2-3 loop forms
* so 3-4 does not
* txn proceeds

ribosome stals on tryptophan codons

allows formation of 2-3 stem loop = transcription proceeds

ribosome moves into 1-2 region

prevents formation of 2-3 step loop, transcription terminates and translation initiated

high levels of tryptophan

* complete leader peptide
* ribosome
* trp codons 1 & 2 on line
* 3 & 4 on loop
* stem loop: transcription termination signal

low levels of tryptophan

* incomplete leader peptide
* ribosome stalls
* alternate stem loops: transcription continues
* trp codons 1 & 4 on line
* codons 2 & 3 on loop
* trp regulated genes at the end

attenuation: general starvation

* no ribosomes
* 1-2 and 304 formed
* txn terminated
* repression occurs

more complicated regulation in eukaryotic cells

areas of control:

* chromatin structure
* transcription activators/enhancers/silencers
* RNA processing/degredation
* RNA interference
* translation/protein modification
* epigenetics

chromatin structure: location of nucleosomes

may occlude binding of transcription factors to promoter, chromatin remodeling proteins may be required

chromatin structure: histone acetylation

destabilize nucleosome structure

chromatin structure: histone methylation

may activate or silence, depends on specific amino acid method

in diagrams, acetylation does what?

loosens positively charged tail

DNA methylation

* on CpG islands (5’ CG 3’)
* Cpg islands often found at promoter sites
* cytosine methylated on carbon 5 (methyl group may sit at major groove)
* methylation associated with inactive genes
* methylation status is heritable

transcription

* mediated by several factors
* proteins bind to enhancers and/or promoters
* several transcription factors needed
* tissue specific enhancers
* response elements coordinate regulation
* Prokaryotes just need sigma factor and RNA polymerase

mRNA processing and stability

* RNA must be processed to mature mRNA
* polymerase A splicing
* how stable is the transcript? will it last months or minutes?

translation produces large T antigen while translation produces a…

small t antigen

translational control

* regulation of mRNA localization and localized translational control are governed by cis-regulatory sequences on the mRNA and trans-acting RNA-binding proteins

protein modification and degradation

* must undergo this process before translation, uses RNA-stabilizing proteins which protect it from degradation


* most important part is addition of stabilizing and signaling factors at the 5’ and 3’ ends of the molecule, and the removal of intervening sequences that don’t specify the appropriate amino acids

epigenetics

* changes in gene expression NOT related to changes in DNA sequence
* alterations in chromatin structure
* methylation patterns in DNA
* DNA methylation associated with histone deacetylation
* regulation by noncoding RNA

epigenetics example “good mother rats”

offspring that received more grooming/licking have a different pattern of DNA methylation (genes involved in stress response are expressed

DNA methylation

* on CpG islands
* 5’ CG 3’
* cytosine is methylated on carbon 5
* methyl group may sit at major groove
* methylation associated with inactive genes
* methylation status is heritable

genomic imprinting

gene expression depends on the parent of origin

Prader-Willi and Angelman syndromes

Prader-Willi

* small, poor sexual development, below average IQ
* develop huge appetites and become obese

Angelman

* uncontrolled laughter, muscle movement, seizures
* developmental delays

Deletion on chromosome 15

* PW: delete father’s copy
* Angelman: delete mother’s copy