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Function and regulation of release factor one in Escherichia coli
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2004. , 60 p.
Keyword [sv]
molekylärgenetik
National Category
Microbiology
Identifiers
URN: urn:nbn:se:su:diva-216ISBN: 91-7265-919-X (print)OAI: oai:DiVA.org:su-216DiVA: diva2:191058
Public defence
2004-09-16, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-08-26 Created: 2004-08-26Bibliographically approved
List of papers
1. A novel mutation in ribosomal protein S4 thataffects the function of a mutated RF1
Open this publication in new window or tab >>A novel mutation in ribosomal protein S4 thataffects the function of a mutated RF1
2000 (English)In: Biochimie, ISSN 0300-9084, Vol. 82, no 8, 683-691 p.Article in journal (Refereed) Published
Abstract [en]

Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 °C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts+; on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 °C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.

Place, publisher, year, edition, pages
Société de chimie biologique, 2000
Keyword
RF1; S4; translational termination; readthrough; stop codon
National Category
Biological Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:su:diva-23269 (URN)10.1016/S0300-9084(00)01160-3 (DOI)
Note
Part of urn:nbn:se:su:diva-216Available from: 2004-08-26 Created: 2004-08-26 Last updated: 2010-01-07Bibliographically approved
2. Effects of two cis-acting mutations on the regulation and expression of release factor one in Eschericchia coli
Open this publication in new window or tab >>Effects of two cis-acting mutations on the regulation and expression of release factor one in Eschericchia coli
2004 (English)In: Biochimie, ISSN 0300-9084, Vol. 86, no 7, 431-438 p.Article in journal (Refereed) Published
Abstract [en]

Together with release factor (RF) 2, RF1 recognises the stop codons and triggers the hydrolysis of the nascent peptide from peptidyl-tRNA during translation termination. prfA, the gene that codes for RF1, is located at 27 min on the Escherichia coli map as the second gene in the hemA-operon. The concentration of RF1 has been shown to increase with increased growth rate, but it is not known where and how this control is exerted. In this study we show that the growth rate regulation of RF1, at least in part, is controlled at PhemA1, one of two promoters preceding the hemA gene. We have also characterised two mutations, asuA1 and asuA2, that are antisuppressors to the tRNA suppressor Su2. Our data indicate that the antisuppressor phenotype is caused by an increased amount of RF1. The asuA2 mutation is a G to an A change just downstream of the –10 region of PhemA1, it leads to a higher concentration of RF1 in the cell and abolishes the growth rate regulation. This indicates that the sequence between the –10 region and the transcription start site is important for growth rate control. The increase in concentration of RF1 caused by asuA1 is most likely at the translational level. The efficiency of translation initiation of prfA is low due to a long distance between the start codon and the Shine-Dalgarno (SD) sequence. The asuA1 mutation creates a new start codon with a more optimal distance to the SD sequence. This leads to an increased expression of RF1, probably due to increased initiation efficiency

Place, publisher, year, edition, pages
Société de chimie biologique, 2004
Keyword
RF1; Promoters; Translation initiation; hemA; Growth rate control
National Category
Biological Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:su:diva-23270 (URN)10.1016/j.biochi.2004.06.009 (DOI)
Note
Part of urn:nbn:se:su:diva-216Available from: 2004-08-26 Created: 2004-08-26 Last updated: 2010-01-07Bibliographically approved
3. Studies of the expression of release factor one in Escherichia coli: PhemA1 and identification of a putative promoter
Open this publication in new window or tab >>Studies of the expression of release factor one in Escherichia coli: PhemA1 and identification of a putative promoter
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23271 (URN)
Note
Part of urn:nbn:se:su:diva-216Available from: 2004-08-26 Created: 2004-08-26 Last updated: 2010-01-13Bibliographically approved

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