Ribosome frameshifting and stalling stimulated by 22 base pair mRNA stem-loop structures Sigrid Stokbro Hess June 2013 Supervisor: Michael Askvad Sørensen Department of Biology Picture ref: http://cronodon.com/BioTech/Ribosomes.html 1 Indholdsfortegnelse 1. Preface ....................................................................................................................................................... 3 2. Abstract ..................................................................................................................................................... 4 3. Resumé ...................................................................................................................................................... 5 4. Introduction ............................................................................................................................................... 6 4.1. Translation ......................................................................................................................................... 6 4.2. -1 programmed frameshift: ............................................................................................................... 6 4.3. Models for programmed -1 frameshift ............................................................................................. 7 4.4. Pseudoknots as frameshifting signals ................................................................................................ 8 4.5. Stem-loop structures as frameshift signals ....................................................................................... 9 4.6. Models for the difference between stem-loops and pseudoknots ................................................. 10 4.7. The results of Tholstrup et al. (2012) .............................................................................................. 11 4.8. My study .......................................................................................................................................... 11 5. Materials and methods ........................................................................................................................... 11 5.1. Strains .............................................................................................................................................. 11 5.2. Making of competent cells .............................................................................................................. 12 5.3. Cloning of the DNA constructs ........................................................................................................ 12 5.4. Construction of plasmid .................................................................................................................. 13 5.5. Examining the sequence of pSSH1 and pSSH2 ................................................................................ 14 5.6. Frame-shift assay ............................................................................................................................. 15 5.7. One dimensional gels ...................................................................................................................... 16 5.8. Nonequilibrium two dimensional gels ............................................................................................. 16 6. Results ..................................................................................................................................................... 17 6.1. Plasmids ........................................................................................................................................... 17 6.2. Frameshifting ................................................................................................................................... 19 6.3. Stalling of the ribosomes ................................................................................................................. 20 7. Discussion ................................................................................................................................................ 22 7.1. Modeling the inserted constructs ................................................................................................... 22 7.2. The inserted constructs were able to stimulate frameshift ............................................................ 24 7.3. The inserted constructs were able to stimulate ribosomal stalling ................................................ 26 8. Conclusion ............................................................................................................................................... 26 9. Future studies: ......................................................................................................................................... 27 10. References ........................................................................................................................................... 28 2 1. Preface This Bachelor study was conducted in the period from February 2013 to June 2013 at the Department of Biology, University of Copenhagen, under the guidance of Ass. Professor Michael Askvad Sørensen. I truly appreciate all the help he provided during the entire process. In addition, I would like to thank Marit Warrer, laboratory technician, for tutoring me in laboratory procedures and helping me with various experiments and Mette Konstad for her constructive criticism during the process of writing this thesis. 3 2. Abstract It has long been established that programmed -1 frameshift plays an important physiological role for some viruses and bacteria. -1 frameshift occurs when the ribosome moves 2 instead of 3 bases during a translational elongation cycle. A frameshift signal placed between two slightly overlapping genes (the second gene in the -1 reading frame with respect to the reading frame of the first gene) ensures a fixed ratio of the two protein products, making programmed frameshift an important regulatory mechanism. A frameshift signal consists of a slippery sequence of 7 nucleotides, a spacer of 6 to 9 nucleotides and an RNA structure preventing a fast ribosomal read-through. The pseudoknot is a well known frameshift signal, but in some genes like the Escherichia Coli dnaX, the frameshift signal is believed to be a stem-loop structure. Despite of extensive research of programmed -1 frameshift, the mechanism behind it is still largely unknown. Recent findings reveal that in addition to promoting frameshift, pseudoknots are also able to stimulate ribosomal stalling. Stalling occurs when a frameshifted ribosome is unable to unwind the pseudoknot and is paused at the frameshift signal indefinitely. In the present study, I examine whether expected stem-loop structures induce frameshift and stalling of the ribosome. The sequences, predicted to form stem-loop structures, were derived from a sequence predicted to form a pseudoknot structure known to stimulate both frameshift and ribosomal stalling. By radioactive labeling the proteins from bacteria, transformed with a reporter plasmid containing the predicted stem-loops, I obtained the frameshift efficiency of the analyzed structures and assessed their stalling ability. The analyzed constructs proved to be able to induce both ribosomal frameshifting and stalling, but theoretical structural modeling of the analyzed constructs, casted doubt about the actual structure of the predicted stem-loops. As the actual structure of the examined constructs is debatable it was not possible to draw a conclusion as to whether a stem-loop structure is sufficient to induce ribosomal frameshift and stalling. 4 3. Resumé Det er et velkendt fænomen at programmeret -1 frameshift spiller en vigtig fysiologisk rolle for nogle vira og bakterier. -1 frameshift forekommer når ribosomet rykker 2 i stedet for 3 baser i løbet af en elongeringscyklus. Et frameshiftsignal placeret mellem to let overlappende gener (det andet gen i -1 læseramme i forhold til det første gens læseramme) sikrer en fast ratio af de to proteinprodukter. Denne funktion gør programmeret frameshifting til en vigtig regulativ mekanisme. Et frameshiftsignal består af en slippery(glat) sekvens, en spacer og en RNA struktur der forhindrer ribosomet i at læse hurtigt igennem signalet. Pseudoknuden er et velkendt frameshiftsignal, men i nogle gener, som f.eks. Escherichia Coli dnaX, mener man at frameshiftsignalet er en stem-loopstruktur. På trods af omfattende forskning på området er mekanismen bag programmeret -1 frameshift stadig stort set ukendt. For nyligt blev det opdaget at, udover at fremkalde frameshift, kan pseudoknuder også stimulere stalling af ribosomer. Stalling opstår når det er umuligt for et frameshiftet ribosom at læse igennem pseudoknuden og det derfor bliver standset ved frameshiftsignalet på ubestemt tid. I dette studie undersøger jeg om forventede stem-loop strukturer stimulerer frameshift og stalling af ribosomet. Sekvenserne, der forventes at danne stem-loop strukturer, er afledt af en sekvens forudsagt til at folde som en pseudoknude, der tidligere har stimuleret frameshift og ribosomal stalling. Ved at radioaktivt mærke proteiner fra bakterier transformeret med plasmider indeholdende de forventede stem-loop strukturer, fandt jeg frameshifteffektiviteten for de forventede stem-loop strukturer og vurderede deres evne til at inducere ribosomal stalling. De analyserede strukturer viste sig at være i stand til både at fremkalde ribosomal frameshift og stalling, men teoretiske strukturelle