TY - JOUR
T1 - Translational control of phage f1 gene expression by differential activities of the gene V, VII, IX and VIII initiation sites
AU - Blumer, Kendall J.
AU - Ivey, Mona R.
AU - Steege, Deborah A.
N1 - Funding Information:
We are indebted to Allen Eckhardt and William Kelley for advice and assistance in purifying the gene V-/I-galactosidase fusion protein, to John Abernethy for it’s N-terminal sequence analysis, and to Gary Hoyle for consultation on Western blot analysis. We also thank G. Vergara for photographic assistance and Michael Zuker for the use of the RNAFLD structure prediction program. This work was supported by grants to D.A.S. from the American Cancer Society (NP367) and from the National Institutes of Health (GM33349). D.A.S. is supported in part by a grant from the National Cancer Institute. K.J.B. was supported by a predoct’oral traineeship from the National Institute of General Medical Sciences (GM07184). M.R.I. is the recipient of a predoctoral fellowship from the National Science Foundation.
PY - 1987/10/5
Y1 - 1987/10/5
N2 - Phage-specific transcription and subsequent RNA processing in Escherichia coli infected with the filamentous phage (f1, M13, fd) generate a pool of abundant and relatively long-lived phage mRNA species encoding the four adjacent genes V, VII, IX and VIII. Yet the products of gene V and gene VIII are synthesized at much higher levels than the gene VII and gene IX proteins. To ask if the translational initiation sites heading these genes show corresponding differences in activity and/or functional properties, we have purified a number of the phage mRNAs from cells infected with f1 and examined them in in vitro initiation reactions. The ribosome binding patterns obtained for the phage mRNA species and for smaller defined RNA fragments containing selected initiator regions reveal a large range in apparent ribosome binding strengths. The gene V and gene VIII sites are recognized efficiently in each mRNA species in which they are present. Gene IX site activity appears to be limited by local mRNA structure: the site has undetectable or low ribosome binding activity in all of the phage mRNA species, but is at least tenfold more active if the RNA sequences required to form a potential hairpin stem-and-loop 15 nucleotides upstream from the initiator AUG have been removed. The gene VII site shows no evidence of interaction with ribosomes in any phage mRNA or RNA fragment tested. The same striking differences in initiation activity were observed in vivo by cloning small f1 DNA fragments containing gene V or gene VII initiation site sequences to drive β-galactosidase synthesis. High levels of a gene V-β-galactosidase fusion protein are initiated at the V site, but no detectable synthesis occurs from the VII site. If the VII site is preceded by all of the information encoding the upstream gene V, however, modest amounts of a fusion protein initiated at the VII site are produced. The overall results, in accord with the observed yields of proteins in the phage-infected cell, provide strong evidence that the properties of these translational initiation sites determine in a significant way the differential expression of phage f1 genes V, VII, IX and VIII.
AB - Phage-specific transcription and subsequent RNA processing in Escherichia coli infected with the filamentous phage (f1, M13, fd) generate a pool of abundant and relatively long-lived phage mRNA species encoding the four adjacent genes V, VII, IX and VIII. Yet the products of gene V and gene VIII are synthesized at much higher levels than the gene VII and gene IX proteins. To ask if the translational initiation sites heading these genes show corresponding differences in activity and/or functional properties, we have purified a number of the phage mRNAs from cells infected with f1 and examined them in in vitro initiation reactions. The ribosome binding patterns obtained for the phage mRNA species and for smaller defined RNA fragments containing selected initiator regions reveal a large range in apparent ribosome binding strengths. The gene V and gene VIII sites are recognized efficiently in each mRNA species in which they are present. Gene IX site activity appears to be limited by local mRNA structure: the site has undetectable or low ribosome binding activity in all of the phage mRNA species, but is at least tenfold more active if the RNA sequences required to form a potential hairpin stem-and-loop 15 nucleotides upstream from the initiator AUG have been removed. The gene VII site shows no evidence of interaction with ribosomes in any phage mRNA or RNA fragment tested. The same striking differences in initiation activity were observed in vivo by cloning small f1 DNA fragments containing gene V or gene VII initiation site sequences to drive β-galactosidase synthesis. High levels of a gene V-β-galactosidase fusion protein are initiated at the V site, but no detectable synthesis occurs from the VII site. If the VII site is preceded by all of the information encoding the upstream gene V, however, modest amounts of a fusion protein initiated at the VII site are produced. The overall results, in accord with the observed yields of proteins in the phage-infected cell, provide strong evidence that the properties of these translational initiation sites determine in a significant way the differential expression of phage f1 genes V, VII, IX and VIII.
UR - http://www.scopus.com/inward/record.url?scp=0023645692&partnerID=8YFLogxK
U2 - 10.1016/0022-2836(87)90557-2
DO - 10.1016/0022-2836(87)90557-2
M3 - Article
C2 - 3441007
AN - SCOPUS:0023645692
SN - 0022-2836
VL - 197
SP - 439
EP - 451
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -