Topics1. Overview of gene expression i. Operons ii. The transcription process iii. The translation process2. Regulation of transcription i. Induction and transcription ii. Positive controls iii. Attenuation i. Global control3. Catabolite repression
1.Overview of gene expression Operons Transcription process Translation
Gene expression The processes that cells and viruses use to regulate the way that the information in genes is turned into gene products. Gene regulation is essential for viruses, prokaryotes and eukaryotes . It increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
Switching Genes On & Off All the cells in our body contain identical set of genes How do cells become so different? Express different subsets of genes Each cell switches genes on or off depending on current need Needs to be strictly controlled expression of a gene in a wrong amount, wrong time or wrong cell type can lead to deleterious phenotype (cell death, cancer)
Gene control in eukaryotes Gene expression in eukaryotes is controlled by a variety of mechanisms: those that prevent transcription those that prevent expression after the protein has been produced.
Regulation Occurs at Many Levels1. Transcriptional control2. Post-transcriptional control3. Transport to cytoplasm4. mRNA stability5. Translational control6. Post-translational control
Gene Control in Prokaryotes Prokaryotes have two levels of gene control. Transcriptional mechanisms control the synthesis of mRNA and translational mechanisms control the synthesis of protein after mRNA has been produced.
Operons Operons are groups of genes that function to produce proteins needed by the cell. Two (2) kinds: 1. Structural genes - code for proteins needed for the normal operation of the cell. For example, they may be proteins needed for the breakdown of sugars. The structural genes are grouped together and a single mRNA molecule is produced during their transcription. 2. Regulator genes - code for proteins that regulate other genes. Operons have not been found in eukaryotes
Transcription DNA is transcribed to make RNA (mRNA, tRNA, and rRNA) Transcription begins when RNA polymerase binds to the promoter sequence Transcription proceeds in the 5 3 direction Transcription stops when it reaches the terminator sequence
Translation mRNA is translated in codons (three nucleotides) Translation of mRNA begins at the start codon: AUG Translation ends at nonsense codons: UAA, UAG, UGA Figure 8.2
The Genetic Code 64 sense codons on mRNA encode the 20 amino acids The genetic code is degenerate tRNA carries the complementary anticodon Figure 8.2
The Process of Translation Components needed to begin translations come together. Figure 8.9
The Process of Translation On the assembled ribosome, at tRNA carrying the first amino acid in paired with the start codon on the mRNA. The place where this firsts tRNA sits is called the p site. A tRNA carrying the second amino acid approaches. Figure 8.9
The Process of Translation The second codon of the mRNA pairs with a tRNA carrying the second amino acids joins to the seconds by a peptide bond. This attaches the polypeptide to the tRNA in the p site. Figure 8.9
The Process of Translation The ribosome moves along the mRNA until the second tRNA is in the p site. The next codon to be translated is brought into the a site. The firsts tRNA now occupies the e site. Figure 8.9
The Process of Translation The second amino acid is paired with the start codon on the mRNA. Is release from the e site. Figure 8.9
The Process of Translation The ribosome continues to move along the mRNA and new amino acids are added to the polypeptide Figure 8.9
The Process of Translation When the ribosome reaches a stop codon, the polypeptide is released. Figure 8.9
The Process of Translation Finally, the last tRNA is released ,and the ribosome comes apart. The released polypeptide forms a new protein. Figure 8.9
RegulationTopics: Induction : i. Induction: lac operon and ii. repression: trp operon Positive and negative controls Attenuation Global controls
Induction and repression Constitutive genes are expressed at a fixed rate Other genes are expressed only as needed such as: Inducible genes Repressible genes
Enzyme induction Metabolites or substrates can turn on inactive genes so that they are transcribed. In the process of enzyme induction, the substrate, or a compound structurally similar to the substrate, evokes the formation of enzyme(s) which are usually involved in the degradation of the substrate.
Inducible enzymes: Enzymes that are synthesized as a result of genes being turned on. Inducer: The substance that activates gene transcription. Inducible enzymes are produced only in response to the presence of a their substrate (produced only when needed). So that energy is not wasted to synthesize unneeded enzymes.
Induction of lac operons The best case of enzyme induction involves the enzymes of lactose degradation in E. coli. Only in the presence of lactose does the bacterium synthesize the enzymes that are necessary to utilize lactose as a carbon and energy source for growth. Two enzymes are required for the initial breakdown of lactose: lactose permease, which actively transports the sugar into the cell, and beta galactosidase, which splits lactose into glucose plus galactose. The genes for these enzymes are contained within the lactose operon (lac operon) in the bacterial chromosome
Mechanism of lac operon The lac operon is an example of an inducible operon because the structural genes are normally inactive. They are activated when lactose is present.
The region of DNA where the repressor protein binds is the operator site. The promoter site is a region of DNA where RNA polymerase can bind. The entire unit (promoter, operator, and genes) is an operon. The operator acts like a switch that can turn several genes on or off at the same time.
In order for E.coli to digest lactose, it requires three types of enzymes A, B and C. Hence it requires gene A, B and C. These are the structural genes. In normal condition, the genes do not function because a repressor protein is active and bound to the DNA preventing transcription.
When lactose is present, it acts as an inducer by binding to the repressor protein, thus preventing it from attaching to the operator. RNA polymerase can then bind to the operator and transcribe the mRNA molecule. Three different proteins are synthesized.
When all of the lactose in the cell has been catabolized, the repressor protein binds to the operator and shuts down the operon. The repressor protein is produced by a regulator gene.
Functional and regulatorycomponents of the lac operon Lac I Regulatory gene that encodes for the lac Repressor protein that is concerned with regulating the synthesis of the structural genes in the operon. Lac I is adjacent to the Promoter site of the operon. An active repressor binds to a specific nucleotide sequence in the operator region and thereby blocks binding of RNAp to the promoter to initiate transcription. The lac repressor is inactivated by lactose, and is active in the absence of lactose. O Operator specific nucleotide sequence on DNA to which an active Repressor binds. P Promoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. (The promoter site of the lac operon is further divided into two regions, an upstream region called the CAP site, and a downstream region consisting of the RNAp interaction site. The CAP site is involved in catabolite repression of the lac operon.). If the Repressor protein binds to the operator, RNAp is prevented from binding with the promoter and initiating transcription. Under these conditions the enzymes concerned with lactose utilization are not synthesized. Lac Z, Y and A Structural Genes in the lac operon. Lac Z encodes for Beta-galactosidase; Lac Y encodes the lactose permease; Lac A encodes a transacetylase whose function is not known. lac lactose, the inducer molecule. When lactose binds to the Repressor protein, the Repressor is inactivated; the operon is depressed; the transcription of the genes for lactose utilization occurs.
Repression It is the regulatory mechanism that inhibits gene expression and decrease the synthesis of enzymes Usually a response to the overabundance of an end- product of a metabolic pathway Repression is mediated by regulatory proteins called repressors Repressors block the ability of RNA polymerase to initiate transcription from the repressed genes
Repressive operon Repressible operons are the opposite of inducible operons. Transcription occurs continuously and the repressor protein must be activated to stop transcription. Example is tryptophan. It is an amino acids required by an E.coli
Repressible operon (trp operon)•Genes that code for proteins that produce tryptophan arecontinuously transcribed. Figure 8.13
If tryptophan is present in the environment E. coli does not need to synthesize it and the tryptophan-synthesizing genes should be turned off. This occurs when tryptophan binds with the repressor protein, activating it.
Unlike the repressor discussed with the lac operon, this repressor will not bind to the DNA unless it is activated by binding with tryptophan. Tryptophan is therefore a co-repressor.
The trp operon encodes the genes for the synthesis of tryptophan. This cluster of genes regulated by a repressor that binds to the operator sequences. The activity of the trp repressor for binding the operator region is enhanced when it binds tryptophan known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell. Expression of the trp operon is also regulated by attenuation.
Structural Repressor GenesInducible Inactive Active (inhibits)OperonsRepressible Active Inactive (inhibitsOperons when activated)
Tryptophan in negative feedback inhibition Tryptophan can inhibit the first enzyme in the synthesis pathway. The presence of high levels of tryptophan inhibits the activity of the enzyme as shown in the biosynthesis pathway below.
Positive & negative controls Most gene expression is controlled at the level of transcription Regulation of gene expression by proteins can be either positive or negative. Regulation in prokaryotes is usually negative while it is positive in eukaryotes.
The trp and lac operons discussed above are examples of negative control because a repressor blocks transcription. In one case (lac operon) the repressor is active and prevents transcription. In the other case (trp) the repressor is inactive and must be activated to prevent transcription.
Structural genes Repressor or regulatorNegative control Inducible operon Inactive Active (inhibits)(an active repressorinhibits Repressible operon Active Inactive (inhibitstranscription) when activated)Positive control (an active regulator Inactive Inactive (promotespromotes transcription) when activated)
Positive control mechanisms require the presence of an activator protein before RNA polymerase will attach. The activator protein itself must be bound to an inducer molecule before it attaches to mRNA.
Attenuation The attenuator plays an important regulatory role in prokaryotic cells because of the absence of the nucleus in prokaryotic organisms. The attenuator refers to a specific regulatory sequence that, when transcribed into RNA, forms hairpin structures to stop transcription when certain conditions are not met
CATABOLITE REPRESSION Many inducible operons are not only controlled by their respective inducers and regulatory genes, but they are also controlled by the level of glucose in the environment. The ability of glucose to control the expression of a number of different inducible operons is called CATABOLITE REPRESSION.
When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. But when glucose levels drop, ATP phosphates are released until at last forming cAMP: ATP --> ADP + Pi --> AMP + Pi --> cAMP cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression.
Catabolite Repression(a) Growth on glucose or lactose alone (b) Growth on glucose and lactose combined During lag time, intracellular cyclic AMP increases, the lac operon is transcribed, more lactose is transported into the cell. Figure 8.14
End of chapter 5: Regulation of gene expression