Gene Regulation II :Gene Regulation II :The Ribosome Strikes Back! The Ribosome Strikes Back!

Mechanisms CoveredMechanisms Covered

• Attenuation Control– Tryptophan Biosynthesis

• Riboswitches– Tryptophan Biosynthesis

• Translational Control– RBS strength– Mechanisms that prevent translation

Attenuation ControlAttenuation Control

Relies on the fact that in Bacteria transcription and translation are coupled.

• They occur at the same time! (Draw Diagram)

• Allows translational machinery to effect transcription

Low Tryptophan Levels:1. Slow translation of leader

peptide from Domain 12. This allows hairpin formation

between Domains 2 and 33. Transcription continues

High Tryptophan Levels:1. Fast translation of leader peptide from

Domain 12. Domain 2 blocked by ribosome3. Hairpin formation between Domains 3

and 4 lead to formation of terminator structure!

Attenuation ControlAttenuation Control

Alternative RNA Folding Dictates Alternative RNA Folding Dictates Termination PropertiesTermination Properties

5’ Region of trp operon transcript

RiboswitchesRiboswitches• Riboswitches are structures in mRNA that

regulate gene expression • up to now only found in bacteria• Riboswitches are bound directly by small ligands• vitamins, such as riboflavin, thiamin and cobalamin• amino acids, such as methionine and lysine• purine nucleotides (adenine, guanine)

• The binding of such ligands affects the secondary structure of mRNA containing the riboswitch and thus exerts a regulatory function

• Riboswitches are probably one of the oldest regulatory systems

Riboswitch StructuresRiboswitch Structures

• All known riboswitches fold into compact RNA secondary structures with a base stem, a central multi-loop and several branching hairpins

Riboswitch Mechanism IRiboswitch Mechanism I

• Riboswitches form a defined three-dimensional conformation capable of specifically binding a low molecular ligand (such as an amino acid, vitamin or nucleotide)

• Binding of the ligand stabilizes one particular three-dimensional conformation of the riboswitch

• If no ligand is bound a different three-dimensional conformation of the riboswitch becomes energetically more favourable and is adopted

• The different conformations (i.e., in absence or presence of ligand) have different functional consequences!

Riboswitch Mechanism IIRiboswitch Mechanism II

Vitreschak et al., (2003). Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet. 20, 44-50.

Note the different secondary structures formed by sequences 1 and 2. When base-paired they become part of a transcription terminator structure!

Gene RegulationGene Regulation

• Mostly performed at the transcription level in bacteria such as E.coli– IE John C’s lecture on gene regulation

• However it is possible to regulate at higher levels– Eg. Translation (RNA -> Protein)– Eg. Post-translational modification (Protein ->

Active Protein)

Translational Control in Translational Control in BacteriaBacteria

• ‘Strength’ of ribosome-binding sites (RBSs); this is especially important in bacterial polycistronic messages where different amounts of proteins need to be synthesized from a single mRNA

Note the different lengths and position of the RBSs!

Translational Control in Translational Control in BacteriaBacteria

• Other examples of translational control• Translational repression occurs when excess

ribosomal proteins bind to their own mRNAs to represses their translation. If there is sufficient rRNA, these proteins will bind to it in preference to the mRNA

• The stringent response and attenuation (trp operon and other amino acid biosynthetic operons) are both negative control mechanisms that operate through the ribosome to reduce transcription

Translational Control in BacteriaTranslational Control in Bacteria

• A final example of translational control:• Riboswitches do not only regulate transcription

but can also control the translation efficiency of an mRNA

• They do this by controlling access to the Ribosome Binding (RBS) sequence

• If the RBS is ‘hidden’, ribosomes will not be recruited at all (or very inefficiently) and thus the mRNA will not be effectively translated

Ribosomes get recruited (via RBS) to mRNA -> efficient translation

Ribosomes cannot get recruited to mRNA -> no translation

Ligand

Controlling Ribosome Recruitment Controlling Ribosome Recruitment through RBS Accessibilitythrough RBS Accessibility