6/28/20161 GENE REGULATION Lac Operon &Trp Operon in Bacteria Salam Pradeep

6/28/20162 Gene Regulation Gene Regulation refers to the processes that cells use to turn the information on genes into gene products. This functional gene product may be RNA or Protein & the majority of known mechanisms regulate protein coding genes. Gene regulation is essential for viruses, prokaryotes & eukaryotes as it increases the versatility & adaptability of an organism by allowing the cell to express protein when needed. The first example of gene regulation system was the lac operon, discovered by Jacob & Monod, in which protein involved in lactose metabolism are expressed by E.coli only in the presence of lactose and absence of glucose.

6/28/20163 June Lac Operon Jacques Monod (born February 9, 1910 – May 31, 1976 in Paris) : A French biologist, Awarded Nobel Prize in Physiology/ Medicine in François Jacob (born 17 June 1920 in Nancy, France) Famous for their work on the Lac operon Jacob F; Monod J (June 1961) "Genetic regulatory mechanisms in the synthesis of proteins". J Mol Biol. 3: 318–56.

6/28/20164 What is Lac Operon Lac Operon is an Operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. Consists of 3 adjacent structural genes, a promoter, an operator, a regulator and a terminator. Regulated by several factors including the availability of glucose and of lactose.

6/28/20165 Structure of the Operon Lac operon consists of 3 structural genes, a promoter, an operator, a regulator and a terminator. They are lacZ, lacY, and lacA. lacZ encodes β-galactosidase (LacZ): An intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes β-galactoside permease (LacY)- a membrane-bound transport protein that pumps lactose into the cell. lacA encodes β-galactoside transacetylase (LacA)- an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides. Only lacZ and lacY appear to be necessary for lactose catabolism.

6/28/20166 Specific control of the lac genes depends on the availability of the substrate lactose to the bacterium. The proteins are not produced by the bacterium when lactose is unavailable as a carbon source. The lac genes are organized into an operon; they are oriented in the same direction immediately adjacent on the chromosome and are co-transcribed into a single polycistronic mRNA molecule. Transcription of all genes starts with the binding of the enzyme RNA polymerase (RNAP).

6/28/20167 Lac Operon Organization 3 structural genes involved in lactose metabolism in E.coli These lac genes are organized into the lac Operon An Operon is a cluster of genes along with an adjacent promoter & operator that control the transcription

6/28/20168 Structural Gene Transcription A Single mRNA is Produced

6/28/20169 Polycistronic mRNA - Because it carries the information for more than one type of protein Lac Z + Lac Y + Lac A + Breaks down lactose into glucose & galactose Transports lactose into the cell Not fully understood

6/28/ Lac Operator  Lac O+ is a short region of DNA that lies partially within the promoter  It interacts with the regulatory protein that controls the transcription of the Operon.

6/28/ Lac Regulatory Lac I + produces an mRNA & synthesized a repressor protein The lac regulatory protein is called a repressor because it keeps RNA polymerase from transcribing the structural gene This repressor protein can bind to the operator of the lac operon

6/28/ In Absence of Lactose In the absence of lactose, lac repressors binds to the operator

6/28/ In Presence of Lactose Small amount of it are converted into Binds Changed its Shape & cannot bind to the promoter

6/28/ Positive Control of Lac Operon

6/28/ Catabolite Repression

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6/28/ Trp Operon Trp Operon is an operon in bacteria which promotes the production of tryptophan when tryptophan isn't present in the environment. First repressible operon to be discovered. If the amino acid is present, then the operon is repressed & biosynthetic enzymes are not produced.

6/28/ Trp Operon Consists of Regulatory & Structural Genes 5 Structural Genes – trpE to trpA (Downstream) of Promoter Region Transcribed as Polycistronic mRNA – Responsible for tryptophan biosynthesis Promoter & Operator region regulates the level of transcription

6/28/ Between the Promoter/Operator & the Structural gene is the Leader Region, trpL Transcribed into an mRNA called – Leader mRNA Contains a short ORF & translated into a leader peptide

6/28/ Leader mRNA contains the Attenuator consisting of 4 regions (1,2,3 &4) that fold & forms 3 different loop secondary structure. 1 & 2 pairing results in transcription pause signal 2 & 3 pairing results in an antitermination signal that allows transcription to continue 3 & 4 pairing results in a termination signal which stops further transcription

6/28/ Since there is no nuclear membrane in bacteria transcription & translation occur at the same time Translation of trpL mRNA proceeds while RNAP transcribe the attenuator mRNA After RNAP has transcribed region 2, regions 1 & 2 pair to form a stem loop structure. This structure causes RNAP to pause & allows the ribosome o catch up to it. Transcription is tightly coupled with translation Ribosome’s position along the leader mRNA plays an important role in the regulation of transcription

6/28/ Trp Starved When the cell is starved for Trp, the amount of Trp-tRNA drops dramatically. In the leader mRNA there are 2 Trp codons in tandem. When the ribosome reaches these codons, it stalls because the amino acid is short supply & the leader peptide cannot be completed. Since the ribosome covers region1 of the attenuator, region2 will pair with region3. This pairing is an antitermination signal, thereby allowing RNAP to proceed & translation follows to synthesize enzymes necessary to produce trp

6/28/ When the cell is not starved for tryptophan, there is enough Trp-tRNA for translation When the ribosome reaches the 2 Trp codons, 2 Trps are added to the leader peptide. The ribosome then continues to the stop codon at the end of region 1 of the leader peptide. This prevents region2 from pairing with 3 Instead region 3 pairs with 4 which is a termination signal fort RNAP

6/28/ The key signal for attenuation is the conc n of Trp-tRNA gene in the cell If there is an abundance of Trp & Trp-tRNA, then a short leader mRNA transcript is synthesized & transcription is then terminated No Tryptophan biosynthesis enzymes are made If the cell is deficient in Trp & Trp-tRNA, then RNAP continues transcription past trpL & through the 5 trp genes. Ribosome translate this polygenic mRNA to produce the enzymes for Tryptophan biosynthesis

6/28/ More slides at