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Gene regulation prokaryote spptx

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  • 1. Different cell types of multicellularorganisms contain the same DNA… …but different cell types synthesize different sets of proteins.
  • 2.  A cell typically expresses only a fraction of its genes, and the different types of cells in multicellular organisms arise because different sets of genes are expressed. Types of gene expression: o Constitutive expression: some genes are essential and necessary for life, and therefore are continuously expressed; these genes are called housekeeping genes. o The expression levels of some genes fluctuate in response to the external signals. Some genes demonstrate higher expression level once being activated (induced). Some genes are repressed and their expression levels are lower (repressed). So how do cells know which kinds of proteins to synthesize? …gene expression can be regulated at many of the steps in the pathway from DNA to RNA to protein.
  • 3. Prokaryotic Gene Regulation The best example of genetic control is thehttp://highered.mc well studied system graw- of milk sugarhill.com/olc/dl/1200 (lactose) inducible 77/bio25.swf catabolism in the human symbiote, Escherichi a coli.
  • 4. Regulation of the lac operon (dual control: repression and promotion) Prokaryotic genes are polycistron systems, i.e. several relevant genes are organized together to form a transcription unit called the operon. The lac operon includes 3 structural genes (lacZ, lacY and lacA) that are transcribed in unison. Located near the lac operon, is the lacI gene regulates the operon by producing the lac repressor protein. Both the regulatory gene and the lac operon itself contain: promoters (Pl and Plac) at which RNA polymerase binds, and terminators at which transcription halts. Plac overlaps with the operator site (O) to which the active form of the repressor protein binds. The operon is transcribed into a single long molecule of mRNA that codes for all three polypeptides.
  • 5. http://highered.mcgraw- hill.com/olc/dl/120080/bio27.swf Transcription of the lac operon is down-regulated through the binding of the lac repressor to the operator. In the absence of lactose, the repressor remains bound to the operator and preventing access of the RNA polymerase to the promoter. Transcription is blocked and the operon is repressed.
  • 6.  In the presence of lactose, the repressor is inactivated form and does not bind to the operator. Thus the RNA polymerase may bind to the promoter and transcribe the structural genes into a single cistronic mRNA.
  • 7.  The isomeric form of lactose that binds to the repressor is allolactose. The lac repressor is an allosteric protein capable of reversible conversion between two alternative forms. In the absence of the effector allolactose, the repressor protein is in the form that binds to the lac operator. In the effector’s presence, the repressor mostly exists in the alternative and inactive state.
  • 8.  Transcription of the lac operon is up-regulated through the binding of the cAMP Receptor Protein (CRP) complex to the promoter. It is an allosteric protein that is inactive in the free form but is activated by binding to cAMP. The CRP-cAMP complex binds the promoter of inducible operons, increasing the affinity of the promoter for RNA polymerase to stimulate transcription. The effects of active CRP on the lac operon: a. The CRP-cAMP complex binds to the CRP recognition site near the promoter region b. RNA polymerase binds to the promoter and transcribes the operon.
  • 9. Together, the lacrepressor and CAP provide a very sensitive responseto the cell’s need to utilize lactose- metabolizing enzymes.
  • 10. http://highered.mcgraw-hill.com/olc/dl/120080/bio26.swf How the trp operon is controlled. The tryptophan repressor cannot bind the operator (which is located within the promoter) unless tryptophan first binds to the repressor. Therefore, in the absence of tryptophan, the promoter is free to function and RNA polymerase transcribes the operon. In the presence of tryptophan, the tryptophan-repressor complex binds tightly to the operator, preventing RNA polymerase from initiating transcription.
  • 11. The binding of tryptophan to the tryptophan repressor protein changes its conformation. This structural change enables this gene regulatory protein to bind tightly to a specific DNA sequence (t he operator), thereby blocking transcription of the genes encoding the enzymes required to produce tryptophan( the Trp operon). The 3-D structure of this bacterial helix-turn-helix protein, as determined by x- ray diffraction with and without tryptophan bound, is illustrated.Tryptophan binding increases the distance between the two recognition helices in the homodimer, allowing the repressor to fit snugly on the operator.
  • 12. Regulation of the trp operon: a "riboswitch" The trp operon includes 5 structural genes (trpE,trpD, trpC, trpB, and trpA) as well as promoter (Ptrp), operator (O), and leader (L) sequences. The structural genes are transcribed and regulatedas a unit. The repressor protein, encoded by the trpRgene is inactive (cannot recognize the operator site), or in the free form when tryptophan is not abundant.
  • 13. The polycistronic mRNA encodes for the enzymes of the tryptophan biosynthetic pathway. When complexed with tryptophan, the repressor is active and binds tightly to the operator, blocking access of RNA polymerase tothe promoter and keeping the operon repressed.
  • 14. Absence of nuclear membrane separates transcription and translation and the ribosomes will bind the nascent message soon after it emerges from the RNA polymerase. The close linkage of the processes can lead to interdependent control mechanisms such as the attenuation controlled by the trp leader sequence. The transcript of the trp operon includes 162 nucleotides upstream of the initiation codon for trpE (the 1st structural gene). This leader mRNA includes a section encoding a leader peptide (or sensor) of 14 amino acids. If tryptophan is present (in moderate amounts), the sensor peptide is easily made and the long trp operon mRNA is NOT completed. If tryptophan is scarce, the leader peptide is not easily made and the full operon is transcribed then translated into tryptophan synthetic enzymes. Two adjacent tryptophan (trp) codons within the leader mRNA sequence are essential in the operons regulation. The leader mRNA contains four regions capable of base pairing in various combinations to form hairpin structures.
  • 15.  Attenuation depends upon the ability of regions 1 and 2 and regions 3 and 4 of the trp leader sequence to base pair and form hairpin secondary structures. A part of the leader mRNA containing regions 3 and 4 and a string of eight Us is called the attenuator. The region 3+4 hairpin structure acts as a transcription termination signal; as soon as it forms, the RNA and the RNA polymerase are released from the DNA.
  • 16. During periods of tryptophan scarcity, a ribosome translating the coding sequence for the leader peptide may stall when it encountersthe two tryptophan (trp) codons because of the shortage of tryptophan- carrying tRNA molecules.
  • 17.  Because a stalled ribosome at this site blocks region 1, a region 1+2 hairpin cannot form and an alternative, region 2+3 hairpin is formed instead. The region 2+3 base pairing prevents formation of the region 3+4 transcription termination hairpin and therefore RNA polymerase can move on to transcribe the entire operon to produce enzymes that will synthesize tryptophan. When tryptophan is readily available, a ribosome can complete translation of the leader peptide without stalling. As it pauses at the stop codon, it blocks region 2, preventing it from base pairing. As a result, the region 3+4 structure forms and terminates transcription near the end of the leader sequence and the structural genes of the operon are not transcribed (nor translated). This is example of a "riboswitch", a mechanism which can control transcription and translation through interactions of substrate molecules with an mRNA.
  • 18. http://highered.mcgraw-hill.com/sites/dl/free/0072437316/120060/ravenanimation.htmlhttp://highered.mcgraw-hill.com/olc/dl/120080/bio27.swfhttp://highered.mcgraw-hill.com/olc/dl/120080/bio31.swfhttp://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf

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