Bacterial Keys to Success• Respond quickly to environmental changes

– Simultaneous transcription and translation

• Avoid wasteful activities by using biochemical and genetic controls– Feedback inhibition of key enzymes– DNA binding proteins to control transcription

• Exchange genes with other organisms to improve adaptation to environment– Conjugation; exchange of plasmids and chromosomal

DNA

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Using DNA is expensive to the cell• Model: protein of 50,000 daltons

– Approximately 500 amino acids, so 500 codons• 500 codons is 1500 RNA bases

• Transcription– Every base added (NTP to NMP) spends 2 phosphates, so

equivalent of 2 ATP– 1500 bases/mRNA = 3000 ATP

• Translation– 1 ATP to move ribosome, 1 to charge tRNA /codon– 2 ATP/ codon = 2 * 500 = 1000 ATP for every single

protein molecule produced.

2

Biosynthesis is expensive3

http://www.chempep.com/ChemPep-amino-acids_clip_image004_0001.gif

Feedback Inhibition

• The first enzyme in the pathway has an allosteric site– The first substrate in the pathway fits into the

active site

– The end product fits into the allosteric site

• Shape change from allosteric binding distorts active site– First step in pathway is inhibited

– More efficient than preventing a later step

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5Biochemical regulation: allosteric enzymes

• Allo = other; steric = space. Many enzymes not only have an active site, but an allosteric site.

• Binding of a molecule there causes a shape change in the enzyme. This affects its function.

6Feedback inhibition of pathways

Genetic Regulation

• Biochemical regulation – Prevents the synthesis of unneeded small molecules

• Genetic regulation– Prevents the synthesis of the enzymes that make the

small molecules.– Results in additional energy savings

• Different view of genotype vs. phenotype– Can the bacterium metabolism the sugar sucrose?

(genotype)– Is the bacterium presently able to metabolize it?

(phenotype)

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8Organization of genes• Many genes work together

– Code for proteins that work in same pathway– Systems control genes together, increasing or decreasing

their transcription as needed by the cell.

• Operon– Genes physically adjacent and regulated together

• Regulon– Genes dispersed but controlled by same proteins

9How Gene activity is controlled

Characteristic of many DNA-binding proteins

Shape change may lead to increased binding to DNA or to decreased binding depending on protein.

Binding to DNA affects amount of transcription.

Binding of small molecules to proteins causes them to change shape

Operon Structure 10

• The Promoter is the site on DNA recognized by RNA polymerase as place to begin transcription.

• Operator is location where regulatory proteins bind.• Promoter and Operator are defined by function.

11Patterns of Regulation

• Control of transcription is through binding of proteins to the DNA

• Negative control– Binding of protein to the DNA prevents transcription

• Positive control– Binding of protein to DNA promotes transcription

• Induction and repression.– In induction, the genes are off until they are needed.– In repression, the genes normally in use are shut off when

no longer needed.

Binding of lac repressor to DNA 12

More lac Repressor Research - Lewis & Lu Labs

13Repressible operons

• Operon codes for enzymes that make a needed amino acid (for example); genes are “on”.– Repressor protein is NOT attached to DNA– Transcription of genes for enzymes needed to make

amino acid is occurring.• The change: amino acid is now available in the

culture medium. Enzymes normally needed for making it are no longer needed.– Amino acid, now abundant in cell, binds to repressor protein

which changes shape, causing it to BIND to operator region of DNA. Transcription is stopped.

• This is also Negative regulation (protein + DNA = off).

14Repression picture

Transcription by RNA polymerase prevented.

15Regulation can be fine tuned

The more of the amino acid present in the cell, the more repressor-amino acid complex is formed; the more likely that transcription will be prevented.

Induction

• Genes normally off are turned on– Example of negative regulation

• Presence of inducer allows gene expression– Inducer binds to repressor which comes off DNA

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17Structure of the Lac operonKEY:P O are the promoter and operator regions. lac Z is the gene for beta-galactosidase. lac Y is the gene for the permease. lac A is the gene for a transacetylase. lac I, on a different part of the DNA, codes for the lac repressor, the protein which can bind to the operator.

18How the lac operon worksWhen lactose is NOT present, the cell does not need the enzymes. The lac repressor, a protein coded for by the lac I gene, binds to the DNA at the operator, preventing transcription.When lactose is present, and the enzymes for using it are needed, lactose binds to the repressor protein, causing it to change shape and come off the operator, allowing RNA polymerase to find the promoter and transcribe.

http://www.med.sc.edu:85/mayer/genreg1.jpg

19Lactose is not actually the inducer

Low basal levels of beta-galactosidase exist in the cell. This converts some lactose to the related allolactose which binds to the lac repressor protein.

Synthetic inducers such as IPTG with a similar structure can take the place of lactose/allolactose for research purposes.

http://www.search.com/reference/Lac_operon

20Glucose is the preferred carbon source

Positive regulation• Presence of lactose is not enough

– In diauxic growth graph, lactose is present from the start. Why isn’t operon induced?

• Presence of glucose prevents positive regulation– NOT the same as inhibiting– Active Cyclic AMP receptor protein (CRP) needed to

bind to DNA to turn ON lactose operon (and others)– Presence of glucose (preferred carbon source) prevents

activation of CRP.

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www.answers.com/.../catabolite-activator-protein

Function of CRP 22

Low glucose:Adenylate cyclase activecAMP abundantcAMP binds to CRPComplex binds to DNAPromotes transcription