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PROKARYOTE GENES: E. COLI LAC OPERON – CHAPTER 13
OPEN GENETICS LECTURES – FALL 2015 PAGE 1
CHAPTER 13 – PROKARYOTE GENES: E. COLI LAC OPERON
Figure 1. Electron micrograph of growing E. coli. Some show the constriction at the location where daughter cells separate. The colouring is false. (Flickr-NIAID-CC BY 2.0)
INTRODUCTION
With most organisms, every cell contains essentially the same genomic sequence. How then do cells develop and function differently from each other? The answer lies in the regulation of gene expression. Only a subset of all the genes is expressed (i.e. are functionally active) in any given cell participating in a particular biological process. Gene expression is regulated at many different steps along the process that converts DNA information into active proteins. In the first stage, transcript abundance can be controlled by regulating the rate of transcription initiation and processing, as well as the degradation of transcripts. In many cases, higher abundance of a gene’s transcripts is correlated with its increased expression. We will focus on transcriptional regulation in E. coli ( Figure 1 ). Be aware, however, that cells also regulate the overall activity of genes in other ways. For example, by controlling the rate of mRNA translation, processing, and degradation, as well as the post- translational modification of proteins and protein complexes.
1. THE LAC OPERON – A MODEL PROKARYOTE
GENE
Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod (see Section 4 on page 4 ). In E. coli, and many other bacteria, genes encoding several different polypeptides may be located in a single transcription unit called an operon. The genes in an operon share the same transcriptional regulation, but are translated individually into separate polypeptides. Most prokaryote genes are not organized as operons, but are transcribed as single polypeptide units. Eukaryotes do not group genes together as operons (an exception is C. elegans and a few other species). 1.1. BASIC LAC OPERON STRUCTURE E. coli encounters many different sugars in its environment. These sugars, such as lactose and glucose , require different enzymes for their metabolism. Three of the enzymes for lactose metabolism are grouped in the lac operon : lacZ ,
CHAPTER 13 – PROKARYOTE GENES: E. COLI LAC OPERON
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lacY , and lacA ( Figure 2 ). LacZ encodes an enzyme called β-galactosidase , which digests lactose into its two constituent sugars: glucose and galactose. lacY is a permease that helps to transfer lactose into the cell. Finally, lacA is a trans-acetylase ; the relevance of which in lactose metabolism is not entirely clear. Transcription of the lac operon normally occurs only when lactose is available for it to digest. Presumably, this avoids wasting energy in the synthesis of enzymes for which no substrate is present. In the lac operon, there is a single mRNA transcript that includes coding sequences for all three enzymes and is called a polycistronic mRNA. A cistron in this context is equivalent to a gene. 1.2. CIS- AND TRANS - REGULATORS In addition to these three protein-coding genes, the lac operon contains several short DNA sequences that do not encode proteins, but instead act as binding sites for proteins involved in transcriptional regulation of the operon. In the lac operon, these sequences are called P (promoter) , O (operator) , and CBS (CAP-binding site). Collectively, sequence elements such as these are called cis - elements because they must be located adjacently to the same piece of DNA in order to perform correctly. On the other hand, elements outside from the target DNA (such as the proteins that bind to these cis - elements) are called trans - regulators because (as diffusible molecules) they do not necessarily need to be encoded on the same piece of DNA as the genes they regulate.
2. NEGATIVE REGULATION – INDUCERS AND
REPRESSORS
2.1. lacI ENCODES AN ALLOSTERICALLY REGULATED REPRESSOR One of the major trans - regulators of the lac operon is encoded by lacI , a gene located just upstream from the lac operon ( Figure 2 ). Four identical molecules of lacI proteins assemble together to form a homotetramer called a repressor ( Figure 3 ). This repressor is trans-acting and binds to two cis- acting operator sequences adjacent to the promoter of the lac operon. Binding of the repressor prevents RNA polymerase from binding to the promoter ( Figure 2 , Figure 5. ). Therefore, the operon is not transcribed when the operator sequence is occupied by a repressor. Figure 3. Structure of lacI homotetramer bound to DNA. (Original-Deyholos- CC BY-NC 3.0) 2.2. THE REPRESSOR ALSO BINDS LACTOSE (ALLOLACTOSE) Besides its ability to bind to specific DNA sequences at the at the operator, another important property of the lacI protein lacI protein is its ability to bind to allolactose. If lactose is lactose is present, β-galactosidase (β-gal) enzymes convert a convert a few of the lactose molecules into allolactose ( Figure 4. ). This allolactose can then bind to the lacI protein. This alters the shape of the protein in a way that prevents it from binding to the operator. Proteins which change their shape and functional properties after binding to a ligand are said to be regulated through an allosteric mechanism. Therefore, in the presence of lactose (which β-gal converts to allolatose), the repressor doesn’t bind the operator sequence and thus RNA polymerase is Figure 2. Diagram of a segment of an E. coli chromosome containing the lac operon, as well as the lacI coding region. The various genes and cis-elements are not drawn to scale. (Original- Deyholos-CC BY-NC 3.0)
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4. THE USE OF MUTANTS TO STUDY THE LAC
OPERON
4.1. SINGLE MUTANTS OF THE LAC OPERON
The lac operon and its regulators were first characterized by studying mutants of E. coli that exhibited various abnormalities in lactose metabolism. Mutations can occur in any of the lacZ , lacY , and lacA genes. Such mutations result in altered protein sequences, and cause non- functional products. These are mutations in the protein coding sequences (non-regulatory). Other mutants can cause the lac operon to be expressed constitutively, meaning the operon was transcribed whether or not lactose was present in the medium. Remember that normally the operon is only transcribed if lactose is present. Such mutants are called constitutive mutants. Constitutive mutants are always on and are unregulated by inducers. These include lacO and lacI genes. 4.2. OPERATOR MUTATIONS The operator locus ( lacO ) - One example is O
- , in which a mutation in an operator sequence reduces or precludes the repressor (the lacI gene product) from recognizing and binding to the operator sequence. Thus, in O-^ mutants, lacZ , lacY , and lacA are expressed whether or not lactose is present. Note that this mutation is cis dominant (only affects the genes on the same chromosome) but not in trans (other DNA molecule). Another common name for a defective operator is O c to refer to its constitutive expression. O-^ and Oc^ are synonyms. Note that constitutively expressed O
mutants may not be maximally expressed, and the extent of the mutation can also affect the level of expression. ( Table 1 ) 4.3. INDUCER MUTATIONS ( lacI LOCUS) The lacI locus has two types of mutations: I-^ and IS. One class of mutant allele for lacI (called I- ) either (1) prevents the production of a repressor polypeptide, or (2) produces a polypeptide that cannot bind to the operator sequence. Note that these two alleles would have different genetic sequences, but the phenotype is the same. Theoretically, we should have better locus names to properly identify specific alleles, but for now we’ll group them under one name. With this form of mutation, the repressor cannot bind and transcription can occur without the presence of inducer (allolactose). This can also be referred to as a “constitutive expresser” of the lac operon: the absence of repressor binding permits transcription. Note that I+^ is dominant over I-. For example, in E. coli strain with _I
Z
Y
A
/F I
Z
Y
A +_ , the lac genes will not be inducible because the I + allele will still Figure 7. Both E. coli strands with genotypes I+O+Z-Y+A+/F I+ O- Z+Y-A+ and ISO+Z+Y+A+/F I- O- Z+Y+A+^ will induce all the lac genes because repressor cannot bind to the O-^ sequence on the F-factor and cannot prevent transcription. (Original-Locke-CC BY-NC 3.0) Table 1. Constitutively expressed Oc^ mutants may not be maximally expressed and have various levels of expression. Level Genotype Explanation 100% lac I-^ Oc^ no repressor 10 - 20% lac I+^ Oc^ repressor fails to bind tightly ~1% P+^ Oc, high glucose basal transcription, constitutive 0% P-^ or Z-^ no transcription
PROKARYOTE GENES: E. COLI LAC OPERON – CHAPTER 13
OPEN GENETICS LECTURES – FALL 2015 PAGE 5
produce functional repressors that bind to operator sequence, preventing transcription. ( Figure 8 ) The other class of mutant alleles for lacI are called I s
. The altered amino sequence of their proteins remove the “allosteric site”. This means the repressor polypeptide cannot bind allolactose – and therefore it cannot change its shape. Even in the presence of lactose, it remains attached to the operator, so the lac Z, lacY and lac A genes cannot be expressed (no RNA polymerase, no protein). This mutant constitutively represses the lac operon whether lactose is present or not. The lac operon is not expressed at all and this mutant is called a “super-repressor”. I s is therefore dominant to both
I
and I
- in trans. Therefore, E. coli strains with the genotypes 1) ISZ+Y+A+/F I+Z+Y-A+^ and 2) ISZ+Y+A+/F I- Z+Y+A+, the lac Z , lac Y and lac A genes will not be inducible ( Figure 9 ). The repressor protein encoded by lacI gene has at least two independent functional domains. This is the reason why it different mutations can give different classes of mutant phenotypes. (Figure 10 ) 4.4. THE F-FACTOR AND TWO lac OPERONS IN A SINGLE CELL – PARTIAL DIPLOID IN E. COLI More can be learned about the regulation of the lac operon when two different copies (each containing mutations) are present in one cell. This can be accomplished by using an F-factor (also known as a plasmid) to carry one copy, while the other is on the genomic E. coli chromosome. This results in a partial diploid in E. coli that contains two independent copies (alleles) of the lac operon and lacI. An F-factor (named so because it creates “fertility” in the cell which contains it) is an episome. This is a self-replicating extrachromosomal piece of DNA: it is outside of the large, circular bacterial chromosome but has its own origin of replication. Jacob and Monod explored the regulation of the lac operon by introducing different genotypes into bacterial cells and noting how they responded in the presence of absence of sugars, namely glucose and lactose using the mutant classes discussed above. Figure 8. E. coli strain with genotype I+Z-Y+A+/F I-Z+Y-A+^ will not produce lac Z, lac Y and lac A products. (Original-Locke-CC BY-NC 3.0) Figure 9. E. coli strains with genotypes 1) ISZ+Y+A+/F I+Z+Y-A+^ and 2) ISZ+Y+A+/F I-Z+Y+A+^ will not produce lac Z, lac Y and lac A products. (Original-Locke-CC BY-NC 3.0) Figure 10. Because the repressor encoded by lac I gene has independent domains, mutations can also occur independently. (Original-Deyholos/Locke-CC BY-NC 3.0)
PROKARYOTE GENES: E. COLI LAC OPERON – CHAPTER 13
OPEN GENETICS LECTURES – FALL 2015 PAGE 7
___________________________________________________________________________
SUMMARY:
- Regulation of gene expression is essential to the normal development and efficient functioning of cells
- Gene expression may be regulated by many mechanisms, including those affecting transcript abundance, protein abundance, and post-translational modifications
- Regulation of transcript abundance may involve controlling the rate of initiation and elongation of transcription, as well as transcript splicing, stability, and turnover
- The rate of initiation of transcription is related to the presence of RNA polymerase and associated proteins at the promoter.
- RNApol may be blocked from the promoter by repressors, or may be recruited or stabilized at the promoter by other proteins including transcription factors
- The lac operon is a classic, fundamental paradigm demonstrating both positive and negative regulation through allosteric effects on trans - factors.
KEY TERMS:
gene expression transcriptional regulation operon lactose glucose lac operon lacZ lacY lacA β-galactosidase permease trans-acetylase P / promoter O / operator CBS CAP-binding site cis-elements trans-regulators lacI homotetramer repressor inducer allosteric cAMP binding protein CAP CAP binding sequence CBS adenylate cyclase constitutive O c / I
- / I s cis dominant cis-acting factors trans dominant trans-acting factors F-factor / episome merozygotes
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STUDY QUESTIONS:
1) List all points in the “central dogma” of gene action the mechanisms that can be used to regulate gene expression. 2) With respect to the expression of β- galactosidase, what would be the phenotype of each of the following strains of E. coli? Use the symbols: +++ Lots of β-galactosidase activity (100%)
- Moderate β-galactosidase activity (10-20%)
- No β-galactosidase activity (0-1%) a) I+, O+, Z+, Y+^ (no glucose, no lactose) b) I+, O+, Z+, Y+^ (no glucose, high lactose) c) I+, O+, Z+, Y+^ (high glucose, no lactose) d) I+, O+, Z+, Y+^ (high glucose, high lactose) e) I+, O+, Z-, Y+^ (no glucose, no lactose) f) I+, O+, Z-, Y+^ (high glucose, high lactose) g) _I
, O
, Z
, Y -_ (high glucose, high lactose) h) I+, Oc, Z+, Y+^ (no glucose, no lactose) i) I+, Oc,Z+, Y+^ (no glucose, high lactose) j) I+, Oc, Z+, Y+^ (high glucose, no lactose) k) I+, Oc, Z+, Y+^ (high glucose, high lactose) l) I-, O+, Z+, Y+^ (no glucose, no lactose) m) I-, O+, Z+, Y+^ (no glucose, high lactose) n) I-, O+, Z+, Y+^ (high glucose, no lactose) o) I-, O+, Z+, Y+^ (high glucose, high lactose) p) Is, O+, Z+, Y+^ (no glucose, no lactose) q) Is, O+, Z+, Y+^ (no glucose, high lactose) r) Is, O+, Z+, Y+^ (high glucose, no lactose) s) Is, O+, Z+, Y+^ (high glucose, high lactose) 3) In the E. coli strains listed below, some genes are present on both the chromosome, and the extrachromosomal F
factor episome. The genotypes of the chromosome and episome are separated by a slash. What will be the β
galactosidase phenotype of these strains? All of the strains are grown in media that lacks glucose. Use the symbols: +++ Lots of β-galactosidase activity (100%)
- Moderate β-galactosidase activity (10-20%)
- No β-galactosidase activity (0-1%) a) I+, O+, Z+, Y+^ / I+, O-, Z-, Y-^ (high lactose) b) I+, O+, Z+, Y+^ / I+, O-, Z-, Y-^ (no lactose) c) _I
, O
, Z
, Y
/ I
, O
, Z
, Y +_ (high lactose) d) I+, O+, Z-, Y+^ / I+, O-, Z+, Y+^ (no lactose) e) _I
, O
, Z
, Y
/ I
, O
, Z
, Y +_ (high lactose) f) _I
, O
, Z
, Y
/ I
, O
, Z
, Y +_ (no lactose) g) I-, O+, Z+, Y+^ / I+, O+, Z-, Y+^ (high lactose) h) _I
, O
, Z
, Y
/ I
, O
, Z
, Y +_ (no lactose) i) _I
, O c , Z
, Y
/ I
, O
, Z
, Y +_ (high lactose) j) I+, Oc, Z+, Y+^ / I+, O+, Z-, Y+^ (no lactose) k) _I
, O
, Z
, Y
/ I
, O c , Z
, Y +_ (high lactose) l) I+, O+, Z-, Y+^ / I+, Oc, Z+, Y+^ (no lactose) m) I+, O+, Z-, Y+^ / Is, O+, Z+, Y+^ (high lactose) n) _I
, O
, Z
, Y
/ I s , O
, Z
, Y +_ (no lactose) o) Is, O+, Z+, Y+^ / I+, O+, Z-, Y+^ (high lactose) p) Is, O+, Z+, Y+^ / I+, O+, Z-, Y+^ (no lactose) 4) What genotypes of E. coli would be most useful in demonstrating that the lacO operator is a cis - acting regulatory factor? 5) What genotypes of E. coli would be useful in demonstrating that the lacI repressor is a trans - acting regulatory factor? 6) What would be the effect of the following loss- of-function mutations on the expression of the lac operon? a) loss-of-function of adenylate cyclase b) loss of DNA binding ability of CAP c) loss of cAMP binding ability of CAP d) mutation of CAP binding site (CBS) cis - element so that CAP could not bind
