7.3.4. Mutation study of Lac operon in merozygotes:
Merozyotes are partial diploid cells. In merozygote we identified that mutation in lac operon is recessive or dominant type. Because dominant (type of mutation cannot be restoring in presence of wild type) and recessive (type of mutation can be restoring in presence of wild type) condition occur only diploid cells. Mutation in lac operon is also cis acting (effect gene expression on same DNA strand to which it occur) or trans acting (effect gene expression on different DNA strand).
Mutation in operator:
In merozygote, if E. coli chromosome is mutated at operator. The Repressor protein cannot bind to it, gene expression occur but exogenote contain normal operator, Repressor protein bind on it and inhibit expression of gene and thus cell survive in lactose containing medium. This type of mutation known as dominant, cis and constitutive.
Mutation in Lac I:
Mutation in Lac I gene may have three type mutation in DBD (I-d), mutation in oligomerization domain (IO) and mutation in ABS (I-s)
When mutation occurs in I-d, gene expression in merozygotes it is suppressed because second chromosome has normal Lac I which encode normal Repressor protein which bind on both operator and suppress the expression. This type of mutation is dominant, trans acting and constitutive.
When mutation occur in IO, In this condition E. coli mutated chromosome Lac I product give mutated Repressor protein And other chromosome give normal Repressor protein since tetramerization occur at DNA than probability of tetramerization at DNA from normal Repressor protein subunit is reducing. Hence probability of gene expression is increased. This type of mutation is trans acting and recessive.
When mutation in ABS I-S , In this condition E. coli mutated chromosome Lac I product give mutated Repressor protein at ABS site. And other chromosome give normal Repressor protein at ABS site. This type of mutation known as trans acting, dominant and uninducible. But if high concentration of allolactose can turn on the gene, hence called super suppressed mutation. Hence probability of gene expression is decreased.
Mutation in promoter:
In merozygote, if E. coli chromosome is mutated at promoter. Thus repressor protein cannot bind to it, gene expression does not occur but exogenote contain normal promoter, then RNA polymerase bind on it and the expression of gene occur and thus cell survive in lactose containing medium. This type of mutation known as recessive and cis acting.
Expression of Lac operon in presence of IPTG : IPTG (isopropyl thio β-galactosidase) is analog of allolactose. It bind with allolactose binding site of repressor protein and induced gene expression to inhibit repressor protein. IPTG not metabolized by β-galactosidase, hence at low concentration it induce gene expression, hence called gratuitous inducer.
Identification of expression of Lac operon:
There are certain method to identified that β-galactosidase is synthesize or not by the bacteria.
- X-gal :- X-gal (5 Bromo, 4-chloro, 3 indole β-galactoside) is substrate for β-galactosidase. After degradation X-gal gives blue color. When E. coli colonies give blue colour in the presence of X-gal. It means cell synthesize β-galactosidase. But if colonies does not produce blue colour, it indicates that β-galactosidase is mutated in bacteria.
- ONPG (ortho-nitrophenyl-β-D-galactoside) is also serving as substrate for β-galactosidase. When β-galactosidase degrade ONPG it give yellow colour but less use in comparison to X-gal because harmful for bacteria.
- There is another β-galactosidase noninducing substrate name Phenyl-β-D-galactopyranoside, which give rise to glucose and galactose upon β -galactosidase action. It is not use as a inducer like allolactose in Lac operon. It is responsible for the growth of bacteria and used in selection of mutants of lacZ.
7.4. Tryptophan operon
Tryptophan amino acid synthesize from chorisimic acid in a multistep pathway. Trp operon consist genes that encode enzymes which synthesize tryptophan from chorismic acid. Trp operon contain five structural gene and a regulatory leader. Structural gene involves in tryptophan synthesis and leader sequence involve in control gene expression.
Control of trp operon expression occur at two level, first is repression (repression control the initiation of transcription) and second is attenuation. Attenuation is called pre mature termination of transcription.
Structure of Trp operon: In Trp operon two promoter; Primary Promoter (P1) and Secondary Promoter (P2). Operator (O) is present within primary promoter. Secondary promoter is a weak promoter which enhance the basal level transcription of trp C, B and Trp A genes.
Trp structural gene are trpE, trpD, trpC, trpB and TrpA these five gene present in downstream of trpL sequence called leader sequence. The length of leader sequence is 162 nucleotide comprises four region 1, 2, 3 and 4, which contain 14 codon ORF including two tryptophan codon at the end of region one. Region 1 and 3 has poly U sequence those are complementary to 2 and 4 contain poly A sequence. These complementary sequence help in regulation of transcription.
Trp operon is negative repressible. Trp R encoded a repressor protein which is present in inactive form but after bind with tryptophan it become active and binds on operator and inhibits expression of trp EDCBA gene.
Some other operon also regulates trp operon such as mtr, aroH, aroL. aroH and aroL gene product play role in chorisimic acid synthesis. Mtr gene product is trp specific permease. Apart from Mtr TraB and aroP also code for trp permease. Thus extracellular trp is uptake by this permease.
7.4.1. Trp operon experssion in the absences or presence of tryptophan :
Tryptophan bind with trpR and trp repressor protein become active after binding of tryptophan. Hence tryptophan acts as co repressor. Trp repressor protein and tryptophan complex bind on operator site and prevent the binding of RNA polymerase. Thus trp operon expression is stop. But if trptophan is absent trp repressor remain in inactive form, And now RNA polymerase easily transcribe the trp structural gene.
7.4.2. Regulation of trp operon through attenuation:
Attenuation is the process of premature mRNA termination in the presence of excess amount of tryptophan. The leader sequence transcript has four complementary sequence and these complementary sequence can form stem loop structure. These sequences of leader sequence called attenuator. In attenuator sequence or leader sequence two adjacent tryptophan codon are present. In prokaryotes transcription is coupled with translation. When nascent leader sequence of mRNA is emerged, ribosome is bound on ribosome binding site of leader sequence and initiate translation of leader sequence.
When tryptophan concentration is high in cell than the trp-tRNA to is found in charged form and ribosome is read the trp codon on nascent leader mRNA. Charge tRNA with tryptophan deliver the tryptophan on growing polypeptide just at the end of translation of leader sequence first and ribosome stalled at a stop codon on region one and mask the region second as a result transcription of third and fourth region occur, both region had complement base. Thus region third and region four form hair pin loop structure. This loop structure terminate the transcription.
Thus transcription of structural gene not takes place. A truncated 140 nucleotides trp L transcript is produced when this premature termination or attenuation occurs.
When Trp is absent the availability of charged tRNA with tryptophan is very less. When ribosome translate emerging leader sequence it reach on trp codon (present in leader sequence region one) and stalled on sequence-one because there are no charged tryptophan tRNA is available.
But transcription is continuous and in this condition the region-one not form hair pin loop with region two because the ribosome is pause at region one of leader sequence. Thus the region one is not free and could not pair with region two however the region two can easily pair with region three. This pairing does not terminate the transcription of structural genes of typtophan operon. Thus no termination loop formation occur so transcription could not stop and structured genes transcribed and translate into enzymes which convert chorisimic acid into tryptophan.