Genetics Chapter 16

levels of gene regulation

1) genes regulated through alteration of DNA or chromatin structure (eukaryotes) - determine which sequences for transcription or the rate of transcription. through DNA methylation and changes in chromatin structure. 2) regulate transcription (pre-mRNA) 3) regulate mRNA processing by adding 5' cap, 3' end cleavage and poly(A) tail, introns removed. deetermine translation, translation rate, amino acid sequence, mRNA stability. 4) regulate mRNA stability. determines amount of proteins made and degradation. 5) translation. whether proteins activate. 6) protein modification

lac operon of E. coli

A negative inducible operon Lactose metabolism - e. coli needs lactose permease to transport lactose into cell. E. coli breaks down into galactose and glucose, catalyzed by beta-galactosidase or convert into allolactose to regulate lactose metabolism. Regulation of the lac operon beta-gal and permease are encoded by adjacent structural genes in lac operon and have promotor. beta-gal is lacZ, permease is lacY, transacetylase is lacA. No lactose, no growth Lactose, but no glucose, increased rate of synthesis = coordinate induction bc Z Y and A are transcribing at the same time by inducer (allolactose) Inducer: allolactose (lacP) lacI: repressor encoding gene lacP: operon promoter lacO: operon operator Repressor at operator = RNA pol binding blocked and no transcription Lactose-> allolactose -> binds to repreessor and repressor releases from operator so RNA pol can bind to promoter and transcribes lac z y and a. Structural genes lacZ: encoding β-galactosidases lacY: encoding permease lacA: encoding transacetylase The repression of the lac operon never completely shuts down transcription.

trp operon of e. coli

A negative repressible operon Controls synthesis of tryptophan Five structural genes trpE, trpD, trpC, trpB, and trpA—five enzymes together convert chorismate to tryptophan. trpE has long 5' UTR transcribed but doesn't encode anything trp promoter upstream = when tryptophan is low, RNA pol binds to promoter and transcribes the 5 structural genes in 1 mRNA, when translates into enzymes to turn chorismate into tryptophan. regulator gene trpR, encodes repressor protein, usually inactive. Has 2 binding sites, one for operator, other for tryptophan (corepressor). Change shape when bind to corepressor so it can bind to operator and overlap promoter. So RNA pol can't bind to promoter and transcription can't happen.

Some operons regulate transcription through attenuation, premature termination of transcription

Attenuation: affects the continuation of transcription, not its initiation. This action terminates the transcription before it reaches the structural genes. long 5' UTR in mRNA transcribed from trp operon contains 4 regions. Region 1 and 2 are comp, 2 and 3, 3 and 4. Allow 5' UTR to fold into two different structures. Attenuator: 1 structure has one hairpin from base pairing with region 1 and 2 and another at region 3 and 4. 3+4 has UUUUU.... bc rho-independent terminator has hairpin with this. A terminator of transcription. Forms when tryptophan is high. Antiterminator: when tryptophan is low, produced between regions 2 and 3. Also has hairpin, but is not followed by UUUU... so doesn't act as terminator. RNA pol keeps going into coding regions and enzymes make tryptophan. Prevents termination. Upstream of region 1, ribosome binding site. Region 1 encodes small protein that has two UGGs that specify tryptophan, so tryptophan is needed for translation of 5' UTR sequence. Four regions of the long 5′ UTR (leader) region of trpE mRNA When tryptophan is high, region 1 binds to region 2, which leads to the binding of region 3 and region 4, terminating transcription prematurely. Four regions of the long 5′ UTR (leader) region of trpE mRNA When tryptophan is low, region 2 binds to region 3, which prevents the binding of region 3 and region 4, and transcription continues.

other sequences control the expression of some bacterial genes

Bacterial enhancers: affects transcription from far away. have binding sites for proteins that increase the rate of transcription at promoters. cause DNA to loop out so transcription activator at enhancer interacts with RNA pol at promoter. position independent so can be moved without affecting ability. Antisense RNA: complementary to targeted partial sequence of mRNA, control gene expression by binding to sequences on mRNA and inhibiting translation. Riboswitches: molecules influence the formation of secondary structures in mRNA. regulator molecules that bind and affect gene expression by influencing formation of structure. found in 5' UTR of mRNA can fold into compact with base stem and hairpins. Sometimes, small molecule binds to riboswitch and stabilizes terminator = premature termination of transcription. Or binding covers ribosome binding site and prevents initation of translation. Otherwise, it eliminates premature terminator or makes ribosome binding site available. Ribozymes: mRNA molecules with catalytic activity RNA mediated repression, produces mRNA that encodes enzyme glutamine:fructose-6-phosphate amidotransferase, helps synthesize small sugar GlcN6P. Without Glc, enzyme produced to make Glc. Enough Glc, binds to ribozyme of mRNA and self-cleavge of mRNA breaks sugar-phosphate backbone of RNA to prevent translation.

A mutation prevents the catabolite activator protein (CAP) from binding to the promoter in the lac operon. What will the effect of this mutation be on the transcription of the operon?

CAP binds to CAP site on lac operon and stimulates RNA pol to bind the lac promoter to increase transcription. So, if mutation prevents CAP from binding, RNA pol will not bind to lac promoter and so there will be less transcription of lac structural genes.

Positive control and catabolite

Catabolite repression: using glucose when available and repressing the metabolite of other sugars. This is a positive control mechanism. The positive effect is activated by catabolite activator protein (CAP). cAMP binds to CAP; together CAP-cAMP complex binds to a site slightly upstream from the lac gene promoter. RNA pol need CAP to be able to bind to promoters. cAMP: adenosine-3′, 5′-cyclic monophosphate important for signalling The concentration of cAMP is inversely proportional to the level of available glucose. RNA pol doesn't like lac promoter, so little lac transcription happens. Low glucose -> more cAMP to bind to CAP -> increase RNA pol binding to lac promoter and increase transcription. CAP has helix-turn-helix motif, causes DNA to bend, which helps bind RNA pol at promoter to initiate transcription

Under which of the following conditions would a lac operon produce the greatest amount of beta galactosidase? The least? Explain your reasoning. Lactose present Absent Condition 1 Yes No Condition 2 No Yes Condition 3 Yes Yes Condition 4 No No

Condition 1 will produce greatest amount of beta galactosidase bc lactose binds to lac repressor -> lac repressor does not like the operator -> RNA pol can access promoter and increase transcription. No glucose allows for increased cAMP -> complex with CAP -> makes RNA pol more efficient at binding to promoter -> more transcription. Condition 2 will produce least amount bc w/o lactose, lac repressor is active and bound operator -> inhibits transcription. Glucose decreases cAMP so CAP and cAMP doesn't make complex so RNA pol won't transcribe lac operon.

DNA binding proteins

Domains: ~ 60-90 amino acids, responsible for binding to DNA, forming hydrogen bonds with DNA and affect expression/regulate genes. Not permanently attached so competition for regulatory sites among DNA binding proteins. Motif: DNA bind proteins in different groups because of these characteristics, within the binding domain, a simple structure that fits into the major groove of the DNA Distinctive types of DNA-binding proteins based on the motif ex: alpha helices fit into DNA double helix helix-turn-helix - 2 alpha helices connected, common in bacteria zinc finger - in eukaryotes, loopof amino acids with Zn ion. leucine zipper - in eukaryotes, "zipper" made of leucine

operon structure

Operon: promoter + additional sequences that control transcription (operator) + structural genes regulates expression of structural genes by controlling transcription structural genes transcribed into 1 mRNA -> enzymes. Enzymes convert precursor into product. 1 promoter, upstream of structural gene. RNA pol binds to promoter and moves downstream to transcribe structural genes Regulator gene: DNA sequence encoding products that affect the operon function but are not part of the operon, has own promotor, transcribedinto mRNA and translated into small protein -> regulator protein bind to operator = where transcription will happen. Operator oberlaps 3' end of promoter and 5' end of structural gene.

What are riboswitches? How do they control gene expression? How do riboswitches differ from RNA-mediated repression?

Riboswitches are regulatory sequences in RNA that can fold compactly into hairpins. At riboswitches, regulatory molecules bind and affect gene expression by changing the structure of mRNA. Can repress/induce/stabilize terminator in mRNA-> early termination. OR can cover the ribosome binding sites of mRNA so translation can't start. RNA mediated repression is different because it uses a ribozyme - catalyzes - and self-cleave the mRNA so translation doesn't happen. Riboswitches affect gene expression while RNA mediated repression self-cleaves the mRNA.

genes and regulatory elements

Structural genes: encoding proteins Regulatory genes: encoding products that interact with other sequences and affect the transcription and translation of these sequences, thus affect gene expression Regulatory genes control may structural genes expression. But constitutive genes that are expressed all the time are not regulated. Regulatory elements: DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences Positive control: processes that stimulate gene expression -> gene regulation Negative control: processes that inhibit gene expression -> gene regulation

Negative and Positive Control: Inducible and Repressible Operons

Two types of transcriptional control Negative - regulator protein is repressor, inhibits transcription and Positive - regulator protein is activator, stimulates transcription Inducible operons: Transcription is usually off and needs to be turned on. Usually for degrading processes, or proteins that break down molecules. Repressible operons: Transcription is normally on and needs to be turned off. Usually for proteins for important processes, used when there is enough. Negative inducible operons: The control at the operator site is negative. Molecule binding is to the operator, inhibiting transcription. Such operons are usually off and need to be turned on, so the transcription is inducible. Operator site overlaps promoter, binding to operator is blocked so RNA pol can't bind = no transcription. Turned on when inducer is present and binds to repressor, inducer alters shape of repressor to prevent from binding to DNA (allosoteric proteins) No inducer = repressor binds to operator and structural genes not transcribed -> no enzymes made. Inducer: small molecule that turns on the transcription. Negative repressible operons: The control at the operator site is negative. But such transcription is usually on and needs to be turned off, so the transcription is repressible. Corepressor present = binds to repressor and activates to prevent transcription = no enzymes made. Corepressor: a small molecule that binds to the repressor and makes it capable of binding to the operator to turn off transcription. Positive inducible operon regulator protein is an activator and binds to DNA at site other than operator and stimulates transcription. Positive repressible operon

Mutations found in 5' UTR of trp operon of E. coli. What is the most likely effect of each mutation on transcription of trp structural genes? a) A mutation that prevents the binding of the ribosome to the 5' end of the mRNA 5' UTR. b) the mutation that changes the trp codons in region 1 of the mRNA 5' UTR into codons for alanine. c) a mutation that creates a STOP codon early in region 1 of the mRNA 5' UTR. d) Deletions in region 2 of the mRNA 5' UTR. e) Deletions in region 3 of mRNA 5' UTR. f) deletions in region 4 of the mRNA 5' UTR.

a) little to no gene transcription b) transcription only when alanine is low c) little to no gene transcription d) little to no transcription e) transcription keeps going f) transcription keeps going g) transcription happens

how does e.coli maintain biochemical flexibility while optimizing energy efficiency?

gene regulation; bacteria carry all genes but only express the ones they need at a time and make needed proteins. With eukaryotes, specialization of genes to certain cell types. ALL organisms need gene regulation.

operon

group of genes that share a common promoter and are transcribed together to make 1 mRNA that encodes several proteins; common in bacteria and archae, not eukaryotes. They control the expression of genes by regulating transcription.

gene regulation

mechanisms and systems that control the expression of genes

lac mutations

partial diploid - two different DNA molecules, ex: e. coli used to find mutations had full bacteria chromosome and extra piece of DNA (F plasmid - has lac operon) -> some parts of lac operon are cis acting (control expression of genes on same DNA) and some are trans acting (control expression of genes on other DNA molecules) Structural gene mutations: affect the structure of the enzymes but not the regulations of their synthesis lacZ+lacY−/lacZ−lacY+ produce fully functional β-galactosidase and permease. it doesn't matter whether it was attached to functional or nonfunctional permease gene or vice versa. Regulator-gene mutations: lacI− leads to constitutive transcription (produced all the time, whether lactose is present or not) of three structural genes bc genes for proteins are in same operon and are regulated by lac repressor protein. lacI+ is dominant over lacI− and is trans acting. A single copy of lacI+ brings about normal regulation of lac operon. superrepressors: lacI^s make defective repressor proteins that can't be activated by repressor proteins bc altered inducer binding site so inducer can't bind to repressor. dominant over lacI+ lacI+lacZ−/lacI−lacZ+ produce fully functional β-galactosidase Operator mutations: lacOc: C = constitutive repressor protein not able to bind to operator. lacOc is dominant over lacO+, which is cis acting. lacI+lacO+Z−/lacI+lacOclacZ+ produce fully functional β-galactosidase constitutively. Promoter mutations lacP−: cis acting, interfere with binding of RNA pol at promoter. so don't produce lac proteins whether lactose is there or not. lacI+lacP−lacZ+/lacI+lacP+lacZ− fails to produce functional β-galactosidase.

remember

regulator gene is trans acting, operator is cis acting

e. coli and trp operon

trp operon encodes 3 enzymes that synthesize tryptophan together. E. coli uses Beta-galactosidase to metabolize lactose, and bacteriophage lambda is a virus that infects E. coli, the same genes control these two things (operon discovered).