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BIO 220

8. Gene Expression

InheritanceAt one time scientists thought proteins were the genetic informationfound in organisms. Later it was found to be DNA.

Nucleic Acids and NucleotidesNucleotides are composed of a nitrogen base, ribose sugar and aphosphate group. A polymer of nucleotides forms a Nucleic Acid orpolynucleotide. 2 forms of nucleic acids found in the cell are DNA andRNA.

DNADeoxyribonucleic acid

4 nucleotides-Adenine (Purine)Guanine (Purine)Cytosine (Pyrimidine)Thymine (Pyrimidine)

Base Pairing between strands of DNAA-T (2 hydrogen bonds)G-C (3 hydrogen bonds)

DNADS polymer of nucleotides in anti-parallel arrangement.

Helical shaped ( helix), right handed helix in the cell.Helix creates Major Groove and Minor Groove in DNA moleculeMany DNA binding proteins read exposed bases in major groove.Some proteins may bind minor groove sequences.

Inside the cell DNA is supercoiled (twisted about its axis).Supercoiling is achieved by DNA Gyrases or Topoisomerases.Topoisomerase II functions by making a DS break and resealing inDNA to result in a twisting of the molecule.Positive Supercoiling occurs when the DNA strand is twisted in thesame direction as the right handed helix.Negative Supercoiling occurs when the twist is in the oppositedirection of the right handed helix.

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DNA may have addition structure depending on the sequenceBent DNA- string of 5 or 6 adenine residues separated by 4 or 5

basesBending of DNA involved in regulatory processes.

Stemloop Structure- created by inverted repeats. Cruciformstructure forms from hydrogen bonding between a single DNA strand.

Structure serves a protein-binding site.

DNA is chemically the same molecule in Prokaryotes and Eukaryotesbut exists in different forms between these groups of organisms.

EukaryotesLinearWrapped around histones (forms nucleosomes)

Found in the nucleusProkaryotes

Circular moleculePackaging lacks histonesFound in cytoplasm

DNA is viewed using a 5 -> 3 orientation.Composed ofcoding and noncoding regions

Coding Regions (Genes)

DNA sequence which encodes the genetic information for a particularprotein or structural RNA.

Noncoding Regions (many examples)Promoters- upstream DNA sequences which activate expression of a

gene. Promoters range from strong to weak. Strongpromoters are positioned in front of genes that are expressedfrequently (example- genes encoding enzymes involved inmetabolism). Weak promoters may activate genes that areexpressed in special situations (example- sporulation genes).

Enhancer Elements- cis acting sequences upstream or downstreamfrom a gene. Transcription Factors bind to such sequencesto affect the strength of a particular promoter.

Origin of Replication- Sequence recognized by replication enzymes.Telomeres- terminal ends of linear DNA molecules.

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How is the genetic information in DNA expressed?

Central DogmaDNA -> mRNA -> Proteins

DNA is the blueprint, i.e. molecular instructions while mRNA contains

a temporary version of the information encoded in the DNA.Proteins are the manifestation of the information containedwithin the gene.

Gene arrangements-One gene for one protein hypothesis (eukaryotes)- Now modified-Regulons (prokaryotes)-Overlapping genes (viral systems)

ribosome wobbleweak ribosome landing pad

TranscriptionThe process of making an RNA version of the information contained in

DNA. RNA is less stable than DNA. As a result RNA transcripts areonly temporary in nature and degrade rapidly in the cell. (Q: What isthe utility of making a temporary version of genetic information?)

Transcription has many steps which can be divided into 3 broadcategories.

InitiationElongationTermination

Steps of transcription

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An enzyme RNA Polymerase (RNA POL) slides along (or reads) DNAuntil it finds a region for which it has high affinity. Enzymestops and binds by forming hydrogen bonds. This region iscalled a Promoter. Promoters are sequences upstream ofthe gene to be transcribed. Promoters contain a Consensus

Sequence, a sequence of nitrogen bases that are commonamong different promoters (example Pribnow box and 35sequence in Eukaryotes). Identification of promoters isoften aided by proteins called Transcription factors.Phosphorylation of RNA Polymerase II in Eukaryotes alsoaffects the activity of the enzyme.

1. In Bacteria RNA POL helps recognize a promoter with assistancefrom many accessory proteins including a well-studied proteincalled Sigma Factor. 70 is the most common form of SigmaFactor in E. coli however 7 different Sigma Factors have so far

been identified. Each Sigma Factor helps RNA Pol identify (andthus transcribe) a different subsets of genes.

In Eukarya the major subclasses of RNA are transcribed bydifferent RNA Polymerases.

RNA Polymerase I- most types of rRNARNA Polymerase II- all mRNARNA Polymerase III- tRNA (and 1 rRNA)

2. RNA Polymerase unwinds a portion of the DNA double helix.

Complimentary ribose nucleotide triphosphate moleculeshybridize to exposed strand.

3. Initiation- RNA POL begins to travel exposed strand andsynthesize the complimentary RNA molecule in a 5 to 3manner. Initiation occurs with the synthesis of a dinucleotide.

4. Elongation- RNA POL polymerizes nucleotide monomers into aRNA polymer. Proteins called Elongation Factors assist in thisstep.

5. As the enzyme moves along the DNA, RNA POL re-zips DNA.

6. Termination- RNA POL encounters a second type ofconsensussequence telling it to stop polymerizing RNA. Termination oftranscription is caused by the DNA sequence.

Different types of termination signalsInverted repeat followed by AAA

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RNA folds into a stemloop structure which derails RNA PolGC rich regions followed by AT rich regionsRho dependent termination

Rho (protein) binds RNAMoves along molecule until it reaches RNA POL

RNA Pol pauses at Rho dependent termination siteRho causes RNA POL to fall off DNA thus endingtranscription.

Transcription produces 3 main types of RNA

mRNAtRNArRNA

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mRNA- Messenger RNAContains an RNA version of the DNA found within a geneThe information in mRNA is directly translated into a protein.mRNA often goes through a maturation process before it is

functional.Precursor mRNA are often called Primary Transcript.Differences exist between prokaryotic and eukaryotic mRNA

including 5 capping, Poly A tail, splicing and the number ofgenes transcribed on a given mRNA molecule.

5 cap-The 5 end of Eukaryotic RNA contains a modified

Guanosine residue which delays degradation of the mRNA transcript(or adds stability).

Poly A tailThe 3 end of Eukaryotic mRNA contains 100-200 residues

of the nucleotide Adenine.Enhances export of mRNA from the nucleusPrevents degradation in cytoplasmMay enhance translation

Splicing-

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Not all segments of Eukaryotic mRNA (and some BacteriaRNA) contain a translatable message.

Introns- untranslated regionsExons- translated regionsIn the nucleus (spliceosome) splicing occurs whereby

introns are removed and exons are linked to form a mature mRNAmolecule.

Spliceosome is comprised of proteins and snRNA.Catalytic activity of spliceosome is due to snRNA, not proteincomponent.Catalytic RNA is also referred to as Ribozymes.

See Figure 7.30, 7.32

Eukaryotic splicing creates a lariat structure out of intron.

Tetrahymena splicing creates a circular intron and a small 15 nt intronfragment. Tetrahymena ribozyme requires a Guanosine residue andMg++ for catalysis.

All ribozymes require metal ions either for catalysis or tertiary folding.Enzymes (or ribozymes) which require metals for catalysis are calledMetaloenzymes.

Other well characterized ribozymes include the Hairpin Ribozyme,Hepatitis Delta Virus Ribozyme and the Hammerhead Ribozyme.

As ribozymes are often contained in introns they can be referred to asSelf-Splicing Introns.

Polycistronic RNAEukaryotic mRNA generally contains 1 gene per mRNA

molecule (monocistronic RNA).Prokaryotes tend to have related genes i.e. genes involved

in the same biochemical pathway close together on their DNA.

As all such genes are required at the same time, the genesare transcribed on a single mRNA, referred to as Polycistronic RNA.

tRNA- Transfer RNA

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Unlike mRNA, tRNA lacks a genetic message but instead acts as astructural element involved in the translation of mRNA sequences.

Short molecule (73-93 nucleotides long)Contains 4 stems and 3 loopsInteracts both with Amino Acids and mRNA

Draw molecule on board

Because tRNA have specific anticodon sequences, a specifictRNA exists for every codon.

100-110 species of tRNA in Eukaryotes60 species of tRNA in Bacteria

Codon- sequence of 3 nucleotides in an mRNA molecule whichcalls for a specific amino acid to be incorporated into a growingprotein.

Genetic Code- Composed of Nucleotide triplets specifying for aparticular amino acid (codon).

Redundant more than one triplet may code for the sameamino acid. Example- ACU, ACC, ACA all encode for Threonine. Allcodons for a particular amino acid may not be used equally; one codonmay be the preferred sequence. For example, in organism X ACU maybe the preferred codon for threonine but in organism Y ACC may bepreferred. Such a situation is called a Codon Bias and must be takeninto account when moving genes from one organism to another.

See Table 7.3

Unconventional Amino AcidsSelenocysteine- the 21st amino acid

UGA is normally a stop codon but sometimes encodes forselenocysteine.

Choice depends on secondary structure of mRNA and flankingsequences.

Selenocysteine is found in both prokaryotic and eukaryotic

organisms.

PyrrolysineUAG is normally a stop codon but sometimes encodes for

pyrrolysine.Choice depends on secondary structure of mRNA and flanking

sequences.First isolated in certain methanogens.

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Charging of tRNA

Covalent linkage of tRNA and Amino Acid (AA)

Involves 2 chemical reactions in cytoplasmCatalyzed by the enzyme Amino Acyl tRNA SynthetaseSpecific Amino acyl tRNA synthase for each codon

AA/combinationEnzyme recognizes the anticodon region of the tRNA and the

amino acid

AA + ATP -> AMP-AA + P-P

tRNA + AMP-AA -> tRNA-AA + AMP + 2 Pi

How are charged tRNA molecules processed according to theinformation in mRNA to make protein?

Involves interaction between mRNA, tRNA and RibosomeKnown as Translation

Ribosome-Complex of proteins and rRNA (Ribosomal RNA)

Consists of 2 subunits- Small subunit and Large subunit

Ribosome Characteristics

Property Prokaryote EukaryoteOverall Size 70s 80sSmall Subunit 30s 40s# Proteins Small su ~21 ~30

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RNA Size 16s 18sLarge Subunit 50s 60s# Proteins Large su ~34 ~50RNA Size 23s, 5s 28s, 5.8s, 5s

Differences in Prokaryotic and Eukaryotic ribosomes have

implications in antibiotic therapy against protein synthesis.

rRNA Contains no genetic message

Acts as a structural and catalytic element in the translationof mRNA into protein.

16s rRNA- helps 30s sub unit identify Shine Delgarno sequence ofmRNA.16s rRNA- physically attaches tRNA via anti-codon loop to ribosome.16s rRNA- participates in recognition of Release Factor proteins.23s rRNA recognized acceptor stem of charged tRNA23s rRNA- catalyzes formation of peptide bonds23s rRNA- aids in translocation

Translation- translating the message in mRNA into proteinInitiation

ElongationTranslocationTermination

Initiation (Prokaryotes)30 s (small subunit) binds to docking site (Shine Delgarno

Sequence) present in mRNA with help of Initiation Factors. 16s rRNA

hybridizes to SD sequence.

tRNAMET binds to 30s s.u. and mRNAThis step requires energy in the form of high energy phosphate bondscontained within GTP.

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tRNAmet is the first AA in eukaryotic protein synthesis (formyl-methionein prokaryotes)

Formyl group or entire amino acid may later be removed in post-translational modifications.

Initiation Factors (proteins) recruit 50s (large) sub unit.At this point the intact ribosome has been formed with tRNAmet in Psite.

Intact ribosome contains 3 sites-A site (acceptor site)P site (peptide site)E site (exit site)

Show drawing on board

tRNAmet (initiation codon) found in what is referred to as the P site(peptide site).

GUG may sometimes serve as alternative initation codon.Formylmethionine initiator tRNA is found in the P site upon completionof the intact ribosome (not valine).

Elongation

With tRNAmet in the P site, the tRNAAA signified by the next codon (RNA

triplet) hybridizes to the mRNA. (For example, lets say the next codonin the mRNA is GGG encoding for Glycine.)

This bonding occurs via hydrogen bonding between the codon in mRNAand the anticodon sequence found in the tRNAgly.

RNA catalyzed condensation reaction creates dipeptide attached totRNAgly-met.

Elongation requires GTP and elongation factors (proteins)

Proteins are synthesized from the amino terminus (or N terminus) tothe carboxy terminus (or Cterminus). Thus covalent linkage occursbetween the C terminus of the nascent peptide and the amino group ofthe next amino acid added to the chain.

At this point in the process the dipeptide linked to tRNAgly-met iscontained in the A site while the tRNA which was linked to methionineis now uncharged but still located in the P site.

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TranslocationRibosome moves along mRNAGTP and elongation factors are required

The dipeptide tRNAgly-met

now occupies the P site of the ribosome.

A site is now vacant, ready for the next tRNA molecule specified by thecodon in the mRNA.

E site contains uncharged tRNA (once linked to methionine).

During subsequent translocation uncharged tRNA once linked tomethionine discharged from the ribosome.

TerminationRelease Factors enters A site and bindsto nonsense or stop

codonRelease Factors hydrolyzes tRNA-protein bond, releasing proteinRibosome dissociates into subunits + mRNA

PolysomesMultiple ribosomes translating same mRNA molecule

SRP- a short peptide leader called a signal sequence may signify thesecretion of a protein or the insertion of that protein into themembrane. A Signal Recognition Particle (SRP) compose ofprotein and RNA binds the signal sequence and facilitates entry of thegrowing peptide into the membrane bound transport protein. This isthe system utilized with Sec translocases (secYEG for example.) Theseproteins require ATP to translocate peptide. Often the SRP recognizedleader sequence is removed upon completion of protein export.

An alternative system called Tat (twin arginine translocase)

protein export system moves a protein across the membrane afterit has been properly folded. Tat protein export uses the Proton Motiveforce to provide the energy required for export.

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Proper Folding-All proteins must be folded in the proper confirmation to be active.Chaperonins may assist in this process.DNA J and DNA Kare ATP dependent chaperonins. They function inthe cell to prevent a protein from folding too quickly 9and possibly

improperly.)

GroEL and GroES are also ATP dependent chaperonins. These proteinsmay help fold proteins that DNA J and DNA K were unable to fold. Theyalso may help re-fold proteins that are partially denatured.

Post translational Modifications-Not all proteins are functional upon the completion of translation. Forexample- signal sequences may be removed, proteins may bephosphorylated, disulfide bridges may form, adenylation atcertain sites or glycosylation may be required to form a functional

protein. Any such change which occurs upon the completion oftranslation is called a Post Translational Modification. Suchcovalent modifications may function to serve as a regulatory step.

For example Glutamine Synthase can be progressively adenylated (upto 9 times). Each adenylation decreases the overall activity of theenzyme. (The enzyme can be activated by removal of the adenylgroups). See Figure 8.6

Inteins and ExteinsProtein splicing has been observed, analogous to mRNA splicing in

Eukaryotes where certain regions are removed (Inreins) and otherregions are covalently linked together (Exteins) to form the matureprotein. DNA Gyrase subunit A in Mycopbacterium leprae undergoessuch processing. (See Figure 8.7)

Removal of Leader SequencesOften the first amino acid (formyl methionine) is removed as part of

post translational modifications. Sometimes a N terminus leadersequence (such as that required for secretion) is removed.

Control of Gene ExpressionOccurs at several levels

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Initiation of transcriptionElongation of transcriptionInitiation of translationPost translational modification

DNA Binding Proteins- MotifsProteins that interact with DNA often have characteristic motifs thatallow the protein to recognize specific sequences in the DNA. MostDNA binding proteins read the Major Groove while some read the MinorGroove of the molecule.

Helix Turn Helix motif contains a stabilizing helix and a recognitionhelix. Often HTH proteins exist as dimmers with interaction occurringbetween two stabilizing helices.

Leucine Zipper proteins are an example of HTH proteins. The twostabilizing helices interact via hydrophobic interactions betweenleucine residues spaced every 7 amino acids.

Zinc Finger motif proteins contain a DNA recognition helix that isstabilized by 2 histadine residues in the helix and 2 cysteine residuesin another part of the protein. The 4 amino acids coordinate to a Zn++

ion. The resulting finger is this kept stable to read DNA.

Regulation of Transcription InitiationNegative Control involves operons and Repressor proteins

Lac Operon- inducibleDiptheria toxin gene- inducibleTrp Operon- repressible

Positive Control- involves Activator proteins

Catabolite Activator Protein (CAP) + cAMP

Operons and RegulonsOperon-Series of genes whose end products function together in

a pathwayLinked by a single promoterContain on/off switch called an OperatorOperator is located between promoter and genes

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Regulon- a series of operons controlled by a single regulatoryprotein

Show diagram on board

Lac Operon-Contains genes to metabolize lactoseLactose- disaccharide of glucose + galactoseNormally off- InducibleRepressor binds operator, prevents transcription (RNA pol cant

proceed)

In the presence of inducer lactose, operon is on.

Lactose binds with repressor, allosteric changes make repressor

inactive.

RNA pol proceeds

(Allosteric regulation refers to a molecule binding a protein in asite other than the proteins active site. Binding results inconfirmational change to active site making it either favorable to bindsubstrate (allosteric activation) or less likely to bind substrate(allosteric inhibition).)

Identification of Lac mutants shed light on this process

TRP Operon

Normally TRP operon is always on, transcribing the enzymes involvedin typtophan biosynthesis. However in the presence of excesstryptophan, operon is shut off. Process is said to be repressible.

Tryptophan binds to repressor causing allostreric changes which allowrepressor to bind operon. Transcription is stopped. Tryptophan acts as

corepressor.

Diptheria ToxinExpression of the gene encoding for the diphtheria toxin is regulatedby Fe3+. Iron binds to repressor protein resulting in an allosteric

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change whereby the repressor is now able to bind DNA and preventtranscription.

In the absence of Fe3+ the repressor is unable to bind the operator andtranscription proceeds.

Why would a toxin be regulated by the presence or absence of iron?

Catabolite RepressionNot all energy sources are equally preferred by the cell, thus certaincatabolic pathways are not always turned on. This is referred to asCatabolite Repression.

The Glucose EffectCatabolite Repression was first observed with cultures grown in mixednutrient broths containing glucose and lactose. Glucose, being thepreferred energy source was utilized first. Upon the depletion ofglucose, growth curves enter a lag phase. Later growth resumes aslactose catabolic pathways are expressed.This pattern of growth is called Diauxic Growth.

Catbolite repression is inactivated by the activator protein, CataboliteActivator Protein CAP, (sometimes called cAMP Binding Protein orCRP).

In the cell, glucose is the preferred energy source. As glucose levelsdrop, so do corresponding levels of ATP. As a result, a secondarymetabolite cAMP (cyclic adenosine monophosphate) begins toaccumulate.

cAMP binds to CAP, resulting in allosteric changes which allow CAPto bind DNA near a promoter region. CAP attracts RNA POL to thepromoter allowing transcription of the gene to occur. In the absence ofCAP-cAMP complex, RNA POL will not bind to the promoter.

Alternate Sigma factors (Regulation at the level ofTranscription)7 different Sigma Factors in E. coli.Amountof each factor controls transcription of various genes.

70 controls transcription of most genes.

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Other sigma factors control genes involved in Nitrogen Assimilation,Stationary Phase, Osmotic Response, Flagella Synthesis.

Regulation at the level of Elongation of TranscriptionExamples- Tryptophan biosynthesis

Tryptophan BiosynthesisThe TRP Operon contains a short Leader Sequence containingtryptophan residues. Translation of the 5 end of an mRNA from the

TRP operon occurs before transcription is complete, or during theElongation phase of transcription.

If the cell is tryptophan starved, translation of the leader sequencecannot occur quickly. Untranslated mRNA located between theribosome and RNA POL folds into a stem-loop structure which allowstranscription to proceed.

If the cell contains excess tryptophan, translation of the leadersequence occurs relatively quickly and the untranslated mRNAbetween the ribosome and RNA POL folds into an alternative stem-loop

structure which derails RNA POL and terminates transcription.

Regulation at the Initiation of TranslationRiboswitchesInteractions between 5 untranslated region and Poly A tail

RiboswitchesmRNA may fold into alternate structures, especially at the 5

untranslated region of the molecule. Such confirmations ofteninvolving the binding of a metabolite (which the biosynthesis for isencoded by the mRNA) to the mRNA. In such instances binding resultsin a confirmation which prevents translation. This is an example ofNegative Feedbackseen at the RNA level. In the absence of themetabolite the mRNA folds into a confirmation which allows translationto proceed.

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Natural Riboswitch Targets:Coenzyme B12Thiamine pyrophosphateFMNS-adenosylmethionine

GuanineAdenineLysine

5 UTR and Poly A tail interactionsThe Poly A tail of mRNA can interact with PABP (Poly A Binding Protein)which in turn interacts with Initiation Factors involved in ribosomeassembly. The entire structure takes on the structure of a closed loopwhich stabilizes the mRNA and increases translational efficiency.

Small RNA regulation of gene expression- SiRNA and MicroRNASiRNA -Antisense RNAInvolves regulatory RNA (as opposed to Regulatory Proteins).

A source ofAntisense RNA is a small gene with identical sequence tothe gene to be regulated, but having its promoter at the 3 end of thegene. When this gene is transcribed, an RNA complimentary to the

mRNA of the regulated gene is expressed. This small RNA, calledAntisense RNA will hybridize to mRNA and form a double strandedRNA complex which is not translatable.

SiRNA (small interfering RNA) forms the basis of a new type of therapyRNAi (RNA interference.) SiRNA is used to study the roles of variousgene products and is also being used in clinical trials as a form ofmedicine, notably SiRNA against macular degeneration.

MicroRNA-These small RNA molecules have recently been shown to affect generegulation, however the mechanisms are not as straightforward asthose with SiRNA. MicroRNA fold into a stem loop structure for stabilitythen interact with various proteins to form a regulatory structure.Some MicroRNA will degrade mRNA while others will merely preventtranslation of the mRNA. A single MicroRNA has the ability to

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regulate expression of whole classes of genes and complexprocesses such as the Cell Cycle.let7 gene encodes for a microRNA molecule that plays a role in celldifferentiation ofC. elegans and allows cells to exit the Cell Cycle.

Cells lacking let7 microRNA cannot exit the cell cycle and proliferatelike tumor cells. Human lung cancer cells lack let7 microRNA.

Signal Transduction (Post translation Modifications)

Signal Transduction is the process by which an external signal results

in intracellular changes/responses. The external signal does not enterthe cell. Often these processes involve 2 proteins and are called theTwo Component System. This system is used in cellular responseto chemotaxis.

Two Component System involvesSensor ProteinResponse Regulator Protein

Sensor ProteinMembrane bound

Binds signal moleculeBinding results in autophosphorylation at Histidine residue.Also called a Sensor Kinase.

Response Regulator ProteinBecomes phosphorylated by Sensor KinaseActivated protein may bind DNA as activator, repressor or serve

other function.

Two Component System becomes terminated by Phosphatase.Phosphatase removes phosphate from Response Regulator Protein.

Metabolic Regulation

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Allostery involves changes in shape of an enzyme that affects itsactivity. Allosteric Effector molecules can bind to an Allosteric siteof the enzyme resulting in a conformational change that eitheractivates or inhibits the enzyme.

Feedback Inhibition results when the end product of a metabolicpathway acts as an Allosteric inhibitor of an enzyme that catalyzes astep earlier in the pathway.

Isoenzymes are different enzymes that catalyze the same reaction.(See Figure 8.5). Aromatic amino acid synthesis involves a precursorstep catalyzed by the enzyme DAHP Synthase. 3 different isoenzymesof DAHP Synthase are allosterically inhibited by different aromaticamino acid end-products. The 3 amino acids (Tyrosine, Phenylalanineand Tryptophan) participate in Concerted Feedback Inhibition toregulate the total cellular activity of DAHP Synthase.

DNA ReplicationQuestion: By what manner does DNA replication occur?

3 theories-ConservativeSemi-conservativeDispersive

Meselson/Stahl data indicates semi-conservative replication

Origin of Replication300 bp section of DNA where initiation of replication occursSequence specificMultiple origins of replicationReplication occurs bidirectionally

Process of DNA Replication1. Origin sequence recognized by Origin Binding Protein.

2. DNA unwinding by Helicase (ATP dependent).3. Unpaired DNA stabilized by Single Strand Binding Proteins.4. Primase generates RNA primer5. DNA Polymerase III (DNA Pol III) synthesizes new DNA strand 5 to

36. RNA primers removed by 5 to 3 exonuclease activity ofDNA POL I7. RNA Primers replaced by 5 to 3 polymerase activity ofDNA POL I8. Nicks are sealed by DNA Ligase

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8. DNA Gyrase (Topoisomerase) supercoils DNA into supercoiledstructure

Features;Replication forks

Okzaki fragmentsTheta StructuresLeading Strand- continuous synthesis, 5->3Lagging Strand- discontinuous synthesis 5->3Replisome

Replisome- A replication factoryProtein:DNA complex which allows fluid replication of leading and

lagging strands.Looping out of lagging strand occurs through DNA interaction

with proteins.Replisome contains Helicases, 2 DNA POL III and Primase!

Rolling Circle replicationSeen in replication of some plasmids, SS DNA viruses, replication ofsatellite RNA.

DS DNA model-1. Nick initiates formation of SS DNA template2. Circular molecule appears to roll away from linear template3. Linear DNA and circular strand replicate separately

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