14. Lac Operon

beginning of the RNA chain is

+1 (first synthesized nucleotide - beginning of the 5’ UTR in mRNA)

upstream (-)

from the transcription start site to the left (promoter - RNAP binding site etc.)

downstram (+)

from the transcription start site to the right

Initiation: binding of RNAP to the promoter

• conformational changes of both promoter and RNAP

• formation of open complex (bubble)

• RNAP synthesizes approx. 10 bases in this phase

Elongation: enhanced grip on template - polymerization

• continues polymerization (faster than first 10 bases)

• unwinds DNA in front

• does little bit of proofreading (lower fidelity)

• anneals DNA behind

• dissociates growing chain from the template

Termination: end of transcription

different ways of finishing transcription

regulatory proteins (trans-factors) that bind to

certain regulatory sequences (cis-elements)

regulation of gene expression through two types of trans-factors

1. activators (“help” RNAP to bind to the promoter) or

2. repressors (prevent RNAP from binding to the promoter)

bacteria have a

• single RNA polymerase

• processive enzyme; transcribes about 50 nt/sec

• composed of 6 subunits with distinct functions

E. coli RNA polymerase 6 subuntis

1. 2 alpha subunits

2. 2 large Beta subunits

3. 1 omega subunit

4. one sigma subunit

two alpha subunits

interaction with regulatory proteins (and DNA - control of initiation frequency)

two large beta subunits

Beta and Beta’ are catalytic subunits - inhibited by rifampicin

one omega subunit

required to restore denatured RNA polymerase in vitro to its fully funcitonal form

one sigma subunit

interaction with promoter - initation

6 subunits = holoenzyme and without omega subunit

core enzyme

e.coli has various sigma subuints

sigma subunit directs the holoenzyme to specific promoters

• each different omega recognizes a specific promotor sequence

• specific gene expression - fast initiation under specific conditions

consensus sequence (s) in general are the

most commonly found nucleotide sequence of DNA or RNA, of related function, found in different locations or organisms

promoters:

sites that are critical for binding of RNAP

promoters are asymmetrical:

direct positioning of RNAP

two consensus sequences in RNAP promoters

1. -10 region (TATA box)

2. -35 region

RNAP binds to dsDNA however,

nucleotide sequence that is on the coding strand (in 5’-3’ orientation) and precedes teh coding sequence for the mRNA is usually given/written

promoter determines

1. which strand will serve as a template

2. starting point for transcription

3. strength of polymerase binding

promoter strength

measure of how strongly RNAP is bound

strong promoter

high frequency of initiation of transcription

weak promoter

low frequency of initiation of transcription

Francois Jacob, Jacques Monod in 1950s found

in bacterial cell, genes are organized in operons

Operon - segment of the genomic DNA which consists of structural genes and adjacent regulatory sequences

all structural genes in the operon code for the proteins necessary in the same metabolic pathway

regulatory gene (s) which are not part of the

operon, are involved as well - code for regulatory proteins

catabolic operons

expression of enzymes involved in catabolism of energy source

biosynthetic operons

expression of enzymes involved in synthesis of energy source

all structural genes are transcribed from the

same promoter sequence

structural genes under influence of

same transcription factors (=regulatory proteins) that recognize the same regulatory elements

all structural genes in an operon are

transcribed as part of the same (single) mRNA - polycistronic mRNA

regulatory genes have

their own promoters

examples of catabolic and biosynthetic operons

1. lactose operon & arabinose operon (catabolic)

2. tryptophan operon (biosynthetic)

in prokaryotes: gene regulation allows a

single cell to adjust its environment

genes encoding catabolic enzymes are “off” when

not needed and only turned “on” when the substrate is present

prokaryotic cells lacks compartments;

therefore transcription and translation are coupled! → rapid response to changes in environment by rapid change in gene expression

example: after sensing lactose in their environment, bacteria make

> 50,000 copies of needed enzymes in 90 seconds

constitutive genes

continuously expressed - required for “ongoing” survival of the cell - house keeping genes (such as those required for protein synthesis, glucose metabolism, utilization etc.)

regulated or inducible genes

expression is regulated (induced or suppressed) as needed, depending on the changes in environment

bacterial genes - organized into operons

1. structural genes most commonly

2. regulatory sequences - binding sites for regulatory proteins

3. regulatory genes (and the proteins they code for) are separate!!

inhibit transcription - negative regulation

regulatory proteins in active state turn “off” the expression of the gene

• binding site - operator

• regulatory proteins could be repressors or aporepressors

• sensitive to changes in cell environment

• recognized by effector molecules

• two types: their binding changes protein conformation

two types of regulatory molecules

1. inducers - inactivate repressor

   • induce transcription (lac operon)

2. co- repressors - activate aporepressors

   • stop transcription (Trp operon)

stimulate transcription - positive regulation

regulatory proteins in active state turn “on” the expression of a gene

• regulatory proteins called activators

• bind to DNA near promoter sequence

• influence affinity of RNAP to stay bound to the promoter

• binding creates conformational changes in DNA near the promotor = allostery

(from closed to open complex)

activators sometimes need

effector molecules

three different e.coli operons → three different types of regulatory proteins and methods of regulating gene expression

1. lactose operon

2. tryptophan operon

3. L-arabinose operon

Lactose operon

• catabolic

• negative regulation

• regulatory protein is repressor; effector = inducer is allolactose

tryptophan operon

• anabolic

• negative regulation

• regulatory protein is aporepressor; effector = co-repressor is tryptophan

L-arabinose operon

• catabolic

• positive and negative regulation

• regulatory protein works as both activator and repressor (regulator) effector = inducer is arabinose

structural genes in lac operon of e.coli

code for enzymes for lactose import and catabolism (lactose-energy source when there is no glucose)

inducible genes in lac operon of e.coli

gene products are made in abundance ONLY when lactose is in the media

lac operon lac Z, lac Y, and lac A

1. enzyme necessary to cleave lactose and to convert lactose into allolactose effector): B-galactosidase coded by lac Z gene

2. enzyme necessary to transport lactose into the cell: permease coded by lac Y

3. Thiogalactoside transacetylase coded by lac A - unknown function

Gene I

codes for repressor protein, has its own promoter

O - operator

element, binding site for repressor

P - promoter

binding site for RNAP

scenario 1: lac operon; lactose is not present in a medium

• RNAP is blocked by repressor

• however, expression of lac operon genes is “leaky”

• RNAP manages to occasionally bind to promoter (it is dynamic system) & proceed with low level of transcription (weak promoter)

  • just enough of permease is made to allow entrance of lactose into the cell if it is present in a medium

scenario 2: lac operon; lactose is getting into medium

• repressor protein comes off

• RNAP binds

• B-galactosilase, permease and transacetylase produced

how did jacob and monod figure out regulation of lac operon

model based on 2 types of mutants

1. constitutive - express all three structural genes from the operon even without lactose

2. noninducible - do not express structural genes from the operon even when lactose is present

apart from mutation int he promoter (p) - RNAP binding site, they found two other loc involved in expression

1. Locus O - mutations lead to constitutive expression

2. Locus I - depending on which part of repressor is affected by mutation the outcome is either constitutive expression or noninducible expression (no expression)

peek at J&M results promoter mutaiton Oc mutation

1. RNA polymerase cannot bind: structural genes are not transcribed, even when lac is present

2. Oc mutation - mutation in operator sequence - constitutive

3. repressor cannot bind - RNA polymerase always transcribes structural genes - even when lac is NOT present

Is - super-repressor mutation - noninducible

lac binding site repressor is mutated in super-repressor - lactose cannot bind and super-repressor cannot change conformation -it is always bound to the operator, structural genes are never transcribed - not even when lac is present

summary: repressor stuck ON the DNA → genes always off

I- mutation - constitutive

operator binding site is mutated in I- repressor - mutated repressor cannot bind to operator - RNAP always transcribes structural genes - even when lac is not present

P-

if RNAP cannot bind to the promoter, structural genes will NEVER be expressed

Oc

dominant over O+ (WT) - bacteria always cleaves lactose; constitutive mutation - structural genes constantly expressed

Is

is dominant over I+ (WT) - bacteria cannot cleave latose; noninducible mutation - structural genes are never expressed

I-

is recessive; I+ (WT) is dominant over I-; constitutive mutation - structural genes constantly expressed

Result: theory that

O is the binding site for regulatory protein I

J & M: proof through complementation testsn

transformation of the bacterial cells with plasmids carrying lac operon (whole or parts)

result is a partially diploid cell = merodiploid

Purpose: artifically create 2 sets of alleles for lac operon genes,

complementation is enabled

• plasmid can carry I gene AND operon or only I gene (and not operon)

different combinations of mutations are possible

also in combination with mutations in structural genes (since its the products of structural genes that are being measured)

J & M: discovered and defined

cis elements and trans factors

Repair fo constitutive mutation lac I- complementation in action

• genomic DNA: lac I- mutation produced repressor cannot bind to operator = constitutive mutation; normal B gal always synthesized

• but plasmid carries normal lac I+ also carries normal Operator (O+) mutated B gal (Z-)

plasmid lac I+ gene codes for a diffusible element that acts in trans by binding to any operator it encounters regardless of chromosomal location

synthesis of normal Beta gal (coded by genomic DNA) will become regulated