Function and regulation of release factor one in Escherichia coli

Sammanfattning: During translation termination the stop codons UAA and UAG are recognised by release factor one (RF1). RF1 binds to the ribosome and mediates the hydrolysis of the nascent peptide from the peptidyl-tRNA. In this process, RF1 interacts directly or indirectly with several ribosomal proteins (r-proteins). To study this interaction in vivo we have used a mutant allele of RF1, prfA1. The mutation causes a temperature sensitive (Ts) phenotype and increased readthrough of the stop codons. A mutant form of r-protein S4 that suppresses this Ts phenotype was isolated. The S4 mutant also increases the readtrough of UAG caused by prfA1. In the mutated S4 allele, rpsD101, Tyr51 is changed to Asp. That change introduces a negatively charged amino acid in a part of S4 that belongs to the positively charged RNA binding surface. This might affect the binding of S4 to the 16S rRNA, to another r-protein, or to RF1, and in that way influence on the function of RF1.One of the few things known about the regulation of RF1 is that expression is decreased with increasing generation time. prfA is the second gene in the hemA-operon located at 27 minutes on the E. coli chromosomal map. We have shown that the growth rate regulation of RF1 is exerted at one of the promoters preceding the hemA gene, PhemA1. The promoter is also growth phase regulated and it is turned off in stationary phase. We have characterised two mutations, asuA1 and asuA2, that increase expression of RF1. The asuA2 mutation is a G to A change one nucleotide downstream of the -10 region of PhemA1. Besides increasing expression of RF1 this mutation also abolishes the growth rate and growth phase regulations we have found. The growth phase regulation is partly dependent on ppGpp. We present two models concerning the effect of ppGpp on PhemA1, and what the asuA2 mutation does to it. The prfA gene has low translation initiation efficiency due to a long spacing between the Shine-Dalgarno (SD) sequence and the start codon. The asuA1 mutation creates a new start codon with a shorter distance to the SD sequence. Most likely this increases the efficiency of translation initiation. That PhemA1 is turned off in stationary phase suggests additional regulation of RF1. Our results indicate a putative promoter for the prfA gene within hemA. This promoter does not seem to be growth rate or growth phase regulated.