The Regulation of Gene Expression in Prokaryotes

 

It may be in the interpretation and analysis of differentiation that the new concepts derived from the study of microorganisms will prove of the greatest value .... Eventually, however, differentiation will have to be studied in differentiated cells. - J. Monod and F. Jacob (1961).

 

I.        Overview

 

A.     The Prokaryote Way of Life

 

1.      Prokaryotes are opportunists. 

2.      Prokaryotes travel light.

3.      Prokaryotes are incredibly efficient.

 

B. Control of Protein Production, figure 20.1, page 571. 

 

1.      Transcriptional regulation –

 

2.      Constitutive –  housekeeping genes -promoters still may have varying effectiveness.

3.      Controlled - leaky – “off “  means transcribed at perhaps 1/10,000th the rate of “on.”

4.      RNA processing –

5.      RNA stability – generally much shorted lived than eukaryotic, but still an opportunity for control.

6.      Translational control –

7.      Post translational control –

C. What is an Operon?

 

1.      A group of gene with a commonality of function that are transcribed as a unit

2.      Preceded by a promoter, generally transcribed as a single polycistronic message

3.      Associated with other regulatory DNA sequences.

4.      These sequences serve as docking sites for regulatory proteins that either up- or down-regulate the rate of transcription.

5.      The regulatory proteins themselves are coded for by other genes outside of the control of the operon promoters and regulators. 

I.        Lac Operon: Inducible production of enzymes needed of a catabolic (breakdown) pathway

 

A.     Structure of the Gene Region, figure 20.5, page 576

 

1.      Structural Genes involved in catabolite metabolism

 

a.       Z – β galactosidase – hydrolyzes lactose to glucose and galactose, figure 20.6, page 576.

b.      Y – galactoside permease – membrane transport molecule bringing lactose into the cell

c.       A – transacetylase – transfers acetyl groups, but no one knows why this is in here.

1.      Operon Promoter - consensus sequence that allows the RNA polymerase to bind. 

 

a.       TATATT (TATA box)

b.      TTGACA

c.       CAP binding site (more later)

1.      Operator – the docking site for the repressor protein, site of the on-off switch. 

2.      Structural Gene for Repressor Protein

 

a.       This gene is called I (referring to the fact that removing it induces transcription,)

b.      It codes for a peptide of 360 amino acids.

c.       Peptide functions as a tetramer

d.      Peptide has some homology to homeodomain proteins (helix-turn-helix).

 

1.      Promoter for Repressor - relatively inefficient.  There are roughly 5 to 20 repressor proteins in the cell at any time.

 

A.     Negative Control – Repression of the Operon

 

1.      Switching

a.       On when the repressor is NOT bound

b.      Off when the repressor is bound.

 

1.      Controlling the Switch

a.       Repressor, by itself, assumes conformation that binds to operator.

b.      Repressor, with allo-lactose bound at an allosteric site, does not bind to the operator.

 

 

1.      Transcribing the Genes

 

a.       RNA polymerase binds to the promoter and begins transcribing.

b.      If the repressor is bound to the promoter, it blocks transcription.

c.       Binding is by weak interactions however, and even in the absence of lactose, some transcription takes place.

d.      If lactose is present, the permease will bring it in.

e.       The minor amount of β-galactosidase will convert some of thse molecules into allo-lactose.

f.        The allo-lactose will bind to the allosteric site of the repressor and snap it open.

g.       The rate of transcription will soar, and the cell will begin translating the message into the enzymes.

 

A.     Positive Control. 

 

1.      Ordinarily the RNA polymerase binds inefficiently to the lac operon promoter.

2.      The polymerase only bind efficiently to the promoter if CAP (catabolite activation protein, product of the crp gene) is also bound to the promoter, figure a, page 581.

3.      CAP only binds to the promoter if it also binds cAMP, figure 20.9, page 579.  CAP also has a DNA binding site and an allosteric site for cAMP.

4.      cAMP is only produced when glucose levels are low.

A.     Help Sessions will ask about effects of mutations on the different genes and gene elements discussed above.

 

I.        Trp Operon: Induction of Anabolic Pathways

A.     Synthesis of Tryptophan

 

 

1.      Structure –related to niacin.

2.      Aromatic amino acid synthesis –branch point between the phe/tyr pathway and the trp path occurs at chorismic acid.

3.      If you’re going to make tryptophan you convert this into anthranilic acid and then three other precursors in sequence before you finally get tryptophan.

4.      Each reaction requires its own enzyme and thus there are five enzymes necessary for making tryptophan and useless for anything else.

5.      So, if you can get tryptophan pre-made you don’t have to do any of this, but if you have to make your own, you need all five or you get nada.

A.     Operon Structure, figure 20.11, page 582

 

1.      Structural genes – E through A – This is the order of function in the pathway

2.      Leader – transcribed at the upstream end of the structural gene and used for control

3.      Promoter – as always

4.      Operator – downstream and overlaps

5.      Repressor protein gene – not contiguous with the operon. (Protein is also a helix-turn-helix, functioning as a dimer, see web reference)

A.     Negative Control: Switching
 

1.      As before, the operon is off when the repressor binds the operator and on when it doesn’t. 

2.      However, the repressor protein binds the operator when it also binds tryptophan at the allosteric site.  If no tryptophan is available, then it does not bind.

A.     Negative Adjustment: Attenuation – prematurely terminates transcription if there’s enough tryptophan available, figure 20.12, page 583

1.      The RNA polymerase begins transcribing the leader sequence upstream of the first (trpE)  gene.

 

2.      This leader has a structure that allows it to fold back on itself in hairpin loops in two options, figure 20.12b.

 

3.      As soon as a length of leader projects from the RNA polymerase, a ribosome attaches to it and begins to translate it.

 

4.      The leader codes for a short (14 amino acid) peptide, two amino acids of are tryptophan (10 and 11).

5.      The two codons that specify trp are found just in front of which segment 1, which is colored blue in the text figure

 

1.      If there is lots of tryptophan around, then there’s plenty of trp~tRNA and the leader is translated rapidly through segments 1 and 2.  They clear the ribosome and pair.

2.      This prevents segment 2 from pairing with segment 3. 

3.      Segment 3 can then pair with segment 4 forming a hairpin followed by a series of uracils.

 

1.      BUT….If there is little tryptophan around, the ribosome gets hung up in front of segment 1 of the leader because it stalls and wait for trp~tRNA to enter the ribosome.

2.      Segment 2 can now pair with segment 3 into a large loose loop that leaves 4 unpaired.

3.      The hairpin structure does not form and transcription continues through the 5 structural genes.

 

1.      This can only work in situation where the ribosome attached to the message just as soon as there’s a free end to hook on to.

2.      Attenuation and repression work together.

 

A.     TRAPS and Attenuation
 

1.      When TRAP binds trp (or other ligand, depending) it activates.

2.      Activated TRAP binds to the 5’ end of messages from the trp operon and the monocistronic message for the trp permease, which is under separate control.

3.      This prevents the ribosome from attaching, resulting in formation of the termination hairpin loop.

 

A.     Riboswitches and Anabolic Control

1.      Cobalamin (vitamin B12) Synthesis

a.       The protein BtuB is part of the B12 permease

b.      If sufficient B12  is available you don’t need to make this.

c.       B12  binds to the leader region of the message.

d.      The leader switches conformation to a form with a twist  that blocks ribosome binding.

1.      Thiamin (Vitamin B1) Synthesis

a.       B1  can bind to the leads of both the polycistronic message with the synthetic enzymes and the message for the permease.

b.      Both message switch conformation and hydrogen bond the ribosome binding region to another part of the leader.

1.      Relevance to Control in Eukaryotes?

a.       Ribozyme switches have been found in excised introns.

b.      They may control the ability of the cell to process its RNA

 

IV. Review: Compare and Contrast Inducible lac Operon with Repressible trp Operon

 

A.     Similarities: Both

 

1.      have structural and repressor protein genes and operator and promoter controlling DNA sequences.

2.      are repressed when repressor bind DNA, induced when it does not

3.      have back-up control

A.     Differences

 

1.      The inducible regulates enzymes metabolizing an occasionally available food source (catabolic pathway). Repressible regulates enzymes that produce a needed product that may be occasionally available ready-made (anabolic pathway).

2.      Inducible repressor binds either the small molecule metabolized (coinducer) or the DNA but not both.  Repressible repressor binds to DNA only with small molecule product (corepressor).

3.      Inducible operons are induced by a substrate; repressible operons are repressed by a product.

4.      Back-up control in lac operon is a positive control necessary for full expression, back-up control in trp operon tightens the negative control.

 

 

References:

 

Barrick, Jeffrey and Ronald R. Breaker. (2007) The Power of Riboswitches.  Scientific American 296 (1): 50 – 57 (January)

 

Trp repressor structure: http://www.nature.com/nature/journal/v317/n6040/abs/317782a0.html