Molecular and Cellular Biology - Term Test 2

Lectures 10-15

how bacteria transcribes genes

uses RNA polymerase holoenzyme

• RNA polymerase core attached to a sigma factor

bacteria and nucleus

bacteria does not have a nucleus

• RNA polymerase and transcription factors exists in the cytoplasm 

polycistronic genes

• many bacteria genes are organized into polycistronic genes

• polycistronic genes produce single mRNA molecule from a single promoter 

• mRNA produced from a polycistronic genes codes for multiple proteins that are translated independently 

• genes that produce mRNA coding for a single protein are called monocistronic 

• turning on one promoter allows bacteria to express multiple related proteins at once

operons 

proteins coded on the same polycistronic genes that work together towards the same goal 

• A-C work together and enable the cell to utilize ‘nutrient Y’

what is transcription 

the major way a cell differentially regulates its genes 

• transcribe a gene more often when its product is in higher demand 

• transcribe a gene in response to changing demands

many gene products are only needed under a certain circumstance

• availability of different nutrients

• responding to external threats

• communicating with other cells

• different types of cells

• different stages of cell cycle

how can bacterial RNA polymerase differentially transcribe genes

using different sigma factors to couple the enzyme to different promoters

transcription factors 

• used in conjunction with RNA polymerase to provide further regulation 

• regulate transpiration by helping or hindering the interaction between RNA polymerase and the promoter 

transcription depressors

• proteins that decrease transcription of genes uses RNA

• in bacteria they bind to a repressor binding site in the genes promoter

• when bound to the promoter, it physically prevents RNA polymerase from binding to it

• this is a negative regulation

  • transcription factor binding to the promoter cause transcription repression

transcription activators

• proteins that increase transcription of genes uses RNA

• in bacteria, they bind to an activator binding site in the genes promoter

• when bound to the promoter, it helps RNA polymerase to bind to promoter

• this is positive regulation

  • transcription factor binding to promoter causes transcription activation

activity of transcription factors

• don’t always bind to DNA

• can be programmed to switched between an active and inactive state

  • only binds to DNA when they are active

• can be regulated using post-translational modification

hypothetical example of transcription factors being  regulated 

• gene A is controlled by a transcription activator, RegX 

• RegX is only active when it is phosphoregualted

• unphosphorylated RegA does not ruined on geneA 

• phosphylation of RegA ‘activates the activator’ resulting in geneA expression 

activities of kinases and phosphates

these activities for RegA may be regulated in response to environmental conditions

• allows the cells to regulate geneA expression response to changing demands survival

small molecules can regulate the activity of transpiration factors

• small, organic molecules are frequently used to regulate activities of transcription factors

• transcription factor has a binding pocked corresponding to a small molecule

  • binding of small molecules alter the shape of the transcription factor, modulating its DNA binding activity 

• therefore, these transcription factors and turned off/on by the presence of  absence of a small molecular 

negative regulation of the tryptophan operon 

• tryptophan operon codes for five proteins used is synthesize Tryptohain 

• E. coli controls trap opener according to concentration of tryptophan in cytoplasm 

• low [tryptophan]: turn on trp openron to produce

• high [tryptophan]: turn off trp operon to stop producing tryptophan 

trp operon promoter

• has a -35 and -10 elements

• operator sequence for TrpR binding in between -35 and -10

• programmed to stop producing tryptophan when there is an abundance of the amino acid

trp repressor (TrpR)

is the negative regulator for the trp operon

• TrpR is not part of the trp operon, it is expressed from a separate, monocistronic gene

TrpR has binding pockets for the amino acid tryptophan 

• tryptophan binding activity TrpR 

• activated TrpR binds to the operator as a homodimer 

low [trytophan] + TrpR inactive

• TrpR does not bind to the operator

• RNA polymerase holoenzyme transcribes from the promoter

• high tryptophan production

high [tryptophan] + TrpR active

• TrpR binds to the operator and blocks the RNA polymerase holoenzyme from binding to promoter

• no more tryptophan prediction

two types of negative regulation 

1. turn on the repressor in presence of the small molecule 

2. turn on the repressor in absence of the small molecules

turn on the repressor in presence of the small molecule

• repressor becomes inactive when small molecules

• example TrpR

turn on the repressor in absence of the small molecules

• repressor becomes inactive when small molecule is added

• example Lacl

two types of positive regulation 

1. turn on the activator in presence of the small molecule 

2. turn on the activator in absence of the small molecules 

turn on the activator in presence of the small molecule

• activator becomes inactive when small molecule is removed

• example: catabolize activator protein, CAP

turn on the activator in activator in absence of the small molecule

• activator becomes inactive when small molecule is added

positive and negative regulation

this is about “what happens to transcription when the transcription factor binds to the promoter” 

small molecules modulating transcription factor for activity

this is about “what happens to the transcription factors DNA binding activity with the presence/absence of small molecules”

positive/negative regulation and small molecules

bacteria combine these regulator mechanisms to control gene expression in response to changing environment/demand

E. coli can use lactose as source of energy 

glucose is the primary sugar that E. coli uses to produce energy 

• glucose undergoes glycolysis and the Krebs cycle, producing energy via substrate level phosphorylation and the electron transport chain 

E. coli can also use lactase as energy 

• lactose is a disaccharide made of galactose and glucose 

lactose metabolism digests lactose into galactose and glucose 

• glucose directly enters glycolysis 

• galactose enters glycolysis by getting metabolized to an intermediate of the pathway (glucose-6-phosphate) 

E.coli and lactose in positive regulation

• E.coli prefers using glucose over lactose

• lactose metabolism uses extra energy to convert lactose into substrate of glycolysis, there is not need to do this if glucose is already available

• turn on lactose metabolism only when lactose is available and glucose is not available

lac operon

• codes for three proteins used to metabolize lactose into glucose nad galactose

• β-galactosidase (coded by E.coli LacZ)

  • hydrolysis lactose to glucose + glactose

  • has about 50% change to produce allo lactose as an intermediate molecule

lactose permeate 

coded by E.coli LacY 

• transports lactose into the environment into cytoplasm 

• co-transports one H+ into cytoplasm with lactose, providing energy for the lactose transportation 

lac operon promoter composition 

weaker version of typical bacterial promoter 

• weaker -35 and -10 promoter elements 

• UP element is absent 

binding site for transcription factors DNA

• three operators for Lac Repressor (Lacl) binding 

• binding site for catabolite activator protein 

lacl represses lac operon

• assume that [lactase] is low

• lac repressor (lacl) is expressed from another gene

• lacl is active in absence of a small one clue, and binds to either

  • operator 3 and operator 1

  • operator 1 and operator 2

• biding of Lacl to the operators bends the DNA in a loop, making it inaccessible for RNA polymerase holoenzyme

  • no transcription from the lac operon

background expression

even when a strong repressor is active, very small amounts of the gene will get transcribed

• no molecular mechanism performs its function with 100% efficiency

background expression of LacZ and LacY 

small amounts of LacZ and LacY are produced even in the presence of Lacl due to the background expression 

• these play critical roles for the activation of lac operon once lactose becomes available 

allolactose represses Lacl

• lactose becomes available in environment

• E. coli has some LacY and LacZ already available due to background expression

• lactose gets transported into cell and some gets converted to allolactose

  • allolactose is a small molecule that inhibits Lacl

overall effect of allolactose

at high [lactose] in environment, Lacl falls off the DNA to make the promoter available for RNA polymerase holoenzyme to bind

CAP activates the lac operon 

• Lac operon promoter is now open for RNA polymerase holoenzyme 

• the enzyme cannot bind stably to the weak promoter by itself 

  • transcription level is still low 

• Catabolite Activator Protein (CAP) is the transcription activator that helps RNA polymerase holoenzyme bind to the promoter to activator transcription 

  • CAP is only active when a small molecule, cAMP, is bond to it 

assume that [environmental glucose] is high 

• E. coli does not want to activate lactose metabolism 

• adenylyl cyclase is an enzyme that converts aTP into cyclic AMP (cAMP) 

• adenylyl cyclase is inhibited when glucose gets imported from the environment 

  • essentially, high [environmental glucose]

  • [cAMP] is low

[environmental glucose] depletes 

• adenylyl cyclase becomes active and begins producing cAMP

• two molecules of cAMP binds to CAP, activating the transcription activator 

• CAP binds to the CAP binding site in lac operon promoter, helping RNA polymerase to bind, activating transcription 

• one of the cCAMP monomer makes physical contact with the RNA polymerase holoenzyme to provide it more support as the enzyme binds to the promoter 

CAP binds to the cAMP binding site as a homodimer

• each monomer binds to one cAMP*

• cAMP binding site located in the middle of the protein

Lac Operon is Controlled by that Lacl Cap

• presence of lactose inactivates Lacl, making the lac operon promoter available for RNA polymerase holoenzyme 

• absence of environmental glucose activates CAP to help RNA polymerase holoenzyme binds to the promoter 

• transcription from lac operon is only turned on when [lactose] is high and [environmental glucose] is low 

  • cytoplasmic glucose (produced by lactose metabolism) does not inhibit the lac operon

• when both [environmental glucose] and [lactose] are high, adenylyl cyclase is inhibited, and CAP remains inactive

• although the lac operon promoter is open, transcription is not activated

• E. coli prioritizes using environmental glucose as energy source over lactose

when [glucose] and [lactose] are low in the environment 

Lacl remains bound to the lac operon promoter 

• CAP will be activated but it cannot activate transcription of lac operon since the promoter is not open 

• in the absence of glucose, E. coli needs to use another sugar 

  • however, go not turn on the lac operon since lactose is also unavailable

Lac operon overview

• systems use two transcription factors and two small molecules to regulate lac operon expression according to the environmental condition

• turn on lactose metabolism only when lactose is available and glucose is not available

regulator network of transcription factors

transcription factors can alter gene expression of a group of genes

• their expression may be regulated by other transcription factors

regulator network may have a cascade of transcription activators/repressors regulating themselves

the network can be regulated by various environmental factors and small molecules that each trigger a species response

transcription factors often regulate multiple promoters

transcription factors can alter gene expression of a group of genes

• all of these genes have the binding site for the transcription factor

tryptophan repressor, TrpR represses at least 5 genes

• one of these genes is the TrpR gene; the gene that codes for TrpR itself

TrpR autoregulates itself in a negative feedback loop

• stop making more TrpR when [tryptophan] is high

• prevents over repression of the Trp operon to make it easier to turn it on once [tryptophan] becomes low

transcription factors often regulate multiple proteins 

catabolite activator protein (CAP) is a global regulator which controls over 180 genes in response to glucose availability 

• metabolism of carbon sources (such as the lac operon)

• iron uptake 

• biofilm formation and antibiotic response 

• quorum sensing, etc. 

high [environmental glucose] down regulates these processes via CAP, this is called glucose catabolite repression 

eukaryotic genes are monocistronic

• almost all eukaryotic protein coding genes are monocistronic

  • produces one protein per gene

• mRNA undergoes additional processing set during eukaryotic transcription

• mRNA modification

  • extensive, covalent modification are made to the initial RNA transcription (pre-mRNA) to produce mature mRAN

• nuclear export

  • export mature mRNA from nucleus to cytoplasm before translation

mRNA modification 

• eukaryotic protein CDS has exons and intron

• once transcribed, the pre-mRNA undergoes three modifications to become a mature mRNA 

• addition of 5’ cap 

• splicing (removal of introns) 

• addition of many adenines at the 3’ end of transcript (3’ poly-adenylation) 

exon

parts of proteins CDS that code for the protein

intron

nucleotide sequences that do not code for protein CDS that are inserted in between exons

• introns must be removed before the protein CDS can be translated into a protein

nuclear export