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Prokaryotic Gene Regulation:

Prokaryotic Gene Regulation:

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Prokaryotic Gene Regulation:

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  1. Prokaryotic Gene Regulation: Lecture 5

  2. Introduction • The two types of transcription regulation control in prokaryotic cells • The lac operon an inducible regulatory feedback system • The tryp operon a repressible regulatory feedback system. • The role of bioinformatics analysis in such systems

  3. Gene Regulation • All “genes” must have some way of regulating their expression (converting the DNA to amino acids) in order to allow them to adopt appropriately to the environment. • In prokaryotic cells the process, owing to the simple nature of the genomic material, is basically controlled “mainly” at the transcription level… • Essentially the molecule “RNA polymerase” must bind to an “exposed“ part of DNA strand called a promoter; it must then move, in the 5’ to 3’ direction, transcribing the DNA of the “gene” to RNA. • The transcription is controlled/ regulated at two stages: • RNA polymerase binds to the DNA strand; • RNA binds DNA transcription begins • RNA is prevented from binding; DNA is not transcribed: gene not expressed. • RNA polymerase moves in the 5’ to 3’ direction: • If the movement if RNA polymerase is blocked transcription is stopped : gene not expressed • Otherwise transcription is completed and the gene is expressed.

  4. Basic types transcriptional control • Inducible: gene is expressed (DNA is converted to RNA) only if the molecule (inducer) is present. • Repressible: if molecule is present gene expression is turned off • In both cases the molecule works via a feedback loop (e.g. a thermostat)

  5. Regulatory loops: transcription Gene • Feedback loop: product of the gene expression loops back (directly/indirectly) and alter the expression of the same gene. • The effect can be either : • positive : “turns on” transcription via an inducer; e.g. [lactose ] • Negative: “turns off” transcription via a repressor e.g. [tryptophan] Gene product

  6. Inducible Prokaryotic regulation • Lactose, a complex sugar (glucose) • In order for E. Coli to use (metabolise) the sugar a gene system referred to as the “lac operon” must produce three enzyme(s) that allow lactose to be utilised by the bacteria. For simplicity we will refer to the combined system as: lactose dehydrogenise (a more detailed description of the enzymes and their functions can be found in Klug p. 310). • The function of this enzyme is to break convert lactose to glucose and increase its absorption into the bacterial cell. • Basically In order to ensure efficient functionality of the “lac operon” system it must have the following properties: • will not be expressed if there is no lactose • will be expressed if there is lactose. • Note a more complete description of the system, involving glucose, can be found in the supplementary notes.

  7. Function of Lac operon • The term operon is the common “gene regulatory system” used in prokaryotic cells and generally a number of genes are regulated as a one. • In the E Coli Lac operon DNA there are: • 1 repressor gene ( • A promoter (where RNA polymerase binds) • A Cis–acting regulatory region (operator) • A leader region (not critical to expression) • 3 structural genes: LacZ, LacY and LacA (refer to here as lactose dehydrogenase) Klug chapter 15

  8. Function of Lac operon Repressor protein • RNA polymerase binds to the promoter region • The repressor gene produces a product “a repressor protein” • This binds to the DNA at the operator region and blocks RNA polymerase moving down the DNA strand. • If lactose is present it alters the repressor protein. • The alter repressor protein is unable to bind to the DNA • RNA polymerase binds to the promoter region and begins transcribing the 3 structural genes. • When lactose levels drop to zero: what happens? RNA polymerase Klug chapter 15

  9. Glucose and the lac operon • Lactose is metabolised into glucose so what happens if glucose is present. • Catabolite-activation protein (CAP): CAP must be present to make RNA polymerase binding efficiently • In the presence of glucose the CAP is altered and prevents RNA polymerase binding to the promoter region and so prevents transcription. Klug chapter 15

  10. A repressible operon • Tryptophan is an essential AA and is normally made (biosynthesised) by E Coli. • If tryptophan is present externally [in sufficient levels] then the biosynthesis stops; • The DNA strand has the same elements as the lactose operon: repressor gene, promoter, operator, leader and genes • In this system the repressor protein is altered by tryptophan and the modified repressor protein now binds to the operator region and blocks RNA polymerase transcribing the genes required to make tryptophan. Klug chapter 15

  11. The tryptophan operon • In addition in the presence of tryptophan there is an additional control mechanism called: • The attenuation regulatory mechanism: • In the sequence prior to structural genes is the attenuator region: • If tryptophan and its gene expression is repressed they still found that transcription was initiated… ; there was “RNA” fragments of leader [L]sequence • Thus altering the repressor protein is not enough to prevent expression. • It seems that tryptophan also binds to the attenuator [A]region and prevents transcription beyond the leader region. Attenuator region Leader region Klug chapter 15

  12. Importance of bioinformatics • Bioinformatics can help with improving our understanding of such regulation by: • Finding potential gene regions and promoter regions since a gene will be in close proximity to a promoter regions. • The prokaryotic sequence normally has specific sequences associated with it and so do genes [begin with AUG/ATG]. [This will be covered in more detail in the lecture “finding genes/promoters” • Prokaryotic systems usually have polycistronic mRNA ; multiple gene sequences in very close proximity (contiguous) or in some cases the may even slightly overlap. (understanding bioinformatics section 9.2) • The gene sequence can be converted into amino acid sequence via universal code • Finally the promoter sequences and gene sequences can be analysed using advanced computational techniques to help understand or suggest how the “operon” works

  13. Exam question • Distinguish, using suitable examples, the difference between inducible and repressible transcription control in prokaryotic organisms. (20 marks) • Discuss, using examples, how analysis of DNA sequences can help our understanding of genes in prokaryotic cells. (10 marks)