Gene Regulation II : The Ribosome Strikes Back!

Mechanisms Covered Attenuation Control –Tryptophan Biosynthesis Riboswitches –Tryptophan Biosynthesis Translational Control –RBS strength –Mechanisms that prevent translation

Attenuation 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 1 2.This allows hairpin formation between Domains 2 and 3 3.Transcription continues High Tryptophan Levels: 1.Fast translation of leader peptide from Domain 1 2.Domain 2 blocked by ribosome 3.Hairpin formation between Domains 3 and 4 lead to formation of terminator structure! Attenuation Control

Alternative RNA Folding Dictates Termination Properties 5’ Region of trp operon transcript

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

Riboswitch 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 II Vitreschak et al., (2003). Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet. 20, Note the different secondary structures formed by sequences 1 and 2. When base-paired they become part of a transcription terminator structure!

Gene 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 Bacteria ‘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 Bacteria 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 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 through RBS Accessibility