TRANSLATION CONTROL OF GENE EXPRESSION Amos Oppenheim, Shoshy Altuvia1, Daniel Kornitzer2, Dinah Teff and Simi Koby Department of Molecular Genetics, Hebrew University-Hadassah Medical School P.O.B. 1172, Jerusalem 91010, Israel
ABSTRACT
The bacteriophage λ cIII gene product is an early regulator of the lysogenic pathway. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrated, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30S ribosomal subunit binding. Translation of the cIII gene is regulated by RNaselll. We have localized the RNaselH responsive element (RRE) to the cIII coding region. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression. The way in which RNaselll participates in this regulation is as yet unknown. KEY WORDS: mRNA structure, translation control, RNaselll This paper was presented at the April 1991 Meeting of the Israel Biochemical Society. Current address: 1 Laboratory of Molecular Biology, Bldg. 37, Room 2E18, NCI, NIH, Bethesda, MD, USA; 2 Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02142, USA
223 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Vol. 2, No. 3, 1991
Translation Control of Gene Expression
INTRODUCTION
Initiation of protein synthesis in prokaryotes takes place by a series of reactions that occurs before the first peptide bond is formed. It proceeds through the formation of an obligate 30S ternary preinitiation complex. This complex is formed with mRNA and initiator fMet-tRNA. The 30S subunit joins to form the 70S initiation complex, which can enter the elongation phase of protein synthesis. These reactions are promoted by three initiation factors and by G T P η, 21. Several studies have indicated that expression of a gene is reduced when the Shine-Dalgarno region and/or the initiation codon are involved in base-pairing interaction /3-7/. Furthermore, alternative m R N A structures, induced by stalling ribosomes, have been proposed to be important in the regulation of expression of certain inducible antibiotic resistance genes /8-10/. Infection of Escherichia coli by bacteroiphage λ may lead to a lytic or lysogenic response / l l / . In the lysogenic response, ell and cIII gene products play a critical role. The ell gene product acts as a positive regulator and activates several phage promoters /12,13/. It is a highly unstable protein with a half-life of 1.5 min /14/. The cIII gene product, a 54-amino-acid-long protein /15/, was found to stabilize the CII protein in vivo, extending its half-life /16, 17/. It was also found to induce the heat shock response, probably through stabilization of σ 32/18/. Phage mutants defective in the cIII gene lysogenize poorly /19/, whereas elevated expression of CIII, caused by point mutations, locks the phage in the lysogenic pathway /20,21/. Analysis of these mutants indicated that the expression of cIII is subject to unique requirements. RESULTS AND DISCUSSION
We have shown that cIII translation is subject to unique requirements /20, 22-24/. We found that the expression of the cIII gene requires sequences located both upstream and downstream of the Shine and Dalgarno sequence and the A U G initiating codon. We proposed a model in which the equilibrium between two alternative mRNA secondary structures, A and B, determines the rate of translation initiation. According to this hypothesis, in structure Β the
224 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Α. Oppenheim, S. Altuvia, D. Kornitzer, D. Teff and S. Koby
Journal of Basic & Clinical Physiology & Pharmacology
mRNA is efficiently translated, whereas in the alternative structure, A, it is unavailable for ribosome binding. Here we summarize the genetic and biochemical evidence supporting this hypothesis. Genetic evidence We have isolated and studied a set of mutants affecting cIII gene activity /20, 23/. All the available cIII mutations can be found in a 100-nucleotide-long sequence around the cIII translation initiation site. The mutations 2 and 17 affect the Shine-Dalgarno sequence and the A U G initiation codon, respectively. Mutations within the coding region can be divided into three categories. The first consists of mutations that dramatically reduce cIII translation. The second category includes mutations which result in elevated expression of cIII. Finally, amber mutation 611, identical to the clllr2 mutation, results in the termination oicIII translation. All the mutations within the cIII coding region cluster in a sequence of 40 nucleotides, which code for the first 14 amino acids of the CIII protein. Surprisingly, no mutation affecting the activity of the CIII protein without affecting its synthesis was isolated by this selection. Our experimental data indicate that the mutations affect cIII expression at the level of translation. The genetic studies are all consistent with a model correlating gene expression with mRNA secondary structure alternatives (Fig. 1). All the mutations located within the coding region which leads to a reduction in cIII translation lower the stability of structure B. Some of these mutations enhance the stability of structure A. On the other hand, all the mutations causing elevated expression of cIII interfere with the formation of structure A. Thus, it seems that one mRNA structure, B, correlates with high-level expression of cIII, whereas structure A correlates with low levels of cIII expression. The mutational data lead us to propose that the cIII mRNA exists in equilibrium between two alternative structures. In one of these structures, B, the mRNA can serve as an efficient template for the translation initiation process, whereas the alternative structure, A, is unavailable for ribosome binding. In the overexpressing mutants, structure Β predominates, whereas in most of the low-level expression mutants, A is favored.
225 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Vol 2 No. 3, 1991
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Translation Control of Gene Expression
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ABSTRACT
The bacteriophage λ cIII gene product is an early regulator of the lysogenic pathway. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrated, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30S ribosomal subunit binding. Translation of the cIII gene is regulated by RNaselll. We have localized the RNaselH responsive element (RRE) to the cIII coding region. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression. The way in which RNaselll participates in this regulation is as yet unknown. KEY WORDS: mRNA structure, translation control, RNaselll This paper was presented at the April 1991 Meeting of the Israel Biochemical Society. Current address: 1 Laboratory of Molecular Biology, Bldg. 37, Room 2E18, NCI, NIH, Bethesda, MD, USA; 2 Whitehead Institute, Nine Cambridge Center, Cambridge, MA 02142, USA
223 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Vol. 2, No. 3, 1991
Translation Control of Gene Expression
INTRODUCTION
Initiation of protein synthesis in prokaryotes takes place by a series of reactions that occurs before the first peptide bond is formed. It proceeds through the formation of an obligate 30S ternary preinitiation complex. This complex is formed with mRNA and initiator fMet-tRNA. The 30S subunit joins to form the 70S initiation complex, which can enter the elongation phase of protein synthesis. These reactions are promoted by three initiation factors and by G T P η, 21. Several studies have indicated that expression of a gene is reduced when the Shine-Dalgarno region and/or the initiation codon are involved in base-pairing interaction /3-7/. Furthermore, alternative m R N A structures, induced by stalling ribosomes, have been proposed to be important in the regulation of expression of certain inducible antibiotic resistance genes /8-10/. Infection of Escherichia coli by bacteroiphage λ may lead to a lytic or lysogenic response / l l / . In the lysogenic response, ell and cIII gene products play a critical role. The ell gene product acts as a positive regulator and activates several phage promoters /12,13/. It is a highly unstable protein with a half-life of 1.5 min /14/. The cIII gene product, a 54-amino-acid-long protein /15/, was found to stabilize the CII protein in vivo, extending its half-life /16, 17/. It was also found to induce the heat shock response, probably through stabilization of σ 32/18/. Phage mutants defective in the cIII gene lysogenize poorly /19/, whereas elevated expression of CIII, caused by point mutations, locks the phage in the lysogenic pathway /20,21/. Analysis of these mutants indicated that the expression of cIII is subject to unique requirements. RESULTS AND DISCUSSION
We have shown that cIII translation is subject to unique requirements /20, 22-24/. We found that the expression of the cIII gene requires sequences located both upstream and downstream of the Shine and Dalgarno sequence and the A U G initiating codon. We proposed a model in which the equilibrium between two alternative mRNA secondary structures, A and B, determines the rate of translation initiation. According to this hypothesis, in structure Β the
224 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Α. Oppenheim, S. Altuvia, D. Kornitzer, D. Teff and S. Koby
Journal of Basic & Clinical Physiology & Pharmacology
mRNA is efficiently translated, whereas in the alternative structure, A, it is unavailable for ribosome binding. Here we summarize the genetic and biochemical evidence supporting this hypothesis. Genetic evidence We have isolated and studied a set of mutants affecting cIII gene activity /20, 23/. All the available cIII mutations can be found in a 100-nucleotide-long sequence around the cIII translation initiation site. The mutations 2 and 17 affect the Shine-Dalgarno sequence and the A U G initiation codon, respectively. Mutations within the coding region can be divided into three categories. The first consists of mutations that dramatically reduce cIII translation. The second category includes mutations which result in elevated expression of cIII. Finally, amber mutation 611, identical to the clllr2 mutation, results in the termination oicIII translation. All the mutations within the cIII coding region cluster in a sequence of 40 nucleotides, which code for the first 14 amino acids of the CIII protein. Surprisingly, no mutation affecting the activity of the CIII protein without affecting its synthesis was isolated by this selection. Our experimental data indicate that the mutations affect cIII expression at the level of translation. The genetic studies are all consistent with a model correlating gene expression with mRNA secondary structure alternatives (Fig. 1). All the mutations located within the coding region which leads to a reduction in cIII translation lower the stability of structure B. Some of these mutations enhance the stability of structure A. On the other hand, all the mutations causing elevated expression of cIII interfere with the formation of structure A. Thus, it seems that one mRNA structure, B, correlates with high-level expression of cIII, whereas structure A correlates with low levels of cIII expression. The mutational data lead us to propose that the cIII mRNA exists in equilibrium between two alternative structures. In one of these structures, B, the mRNA can serve as an efficient template for the translation initiation process, whereas the alternative structure, A, is unavailable for ribosome binding. In the overexpressing mutants, structure Β predominates, whereas in most of the low-level expression mutants, A is favored.
225 Brought to you by | University of Arizona Authenticated Download Date | 7/16/15 7:46 AM
Vol 2 No. 3, 1991
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Translation Control of Gene Expression
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