PMID- 10490635
OWN - NLM
STAT- MEDLINE
DCOM- 20000203
LR  - 20210526
IS  - 0270-7306 (Print)
IS  - 1098-5549 (Electronic)
IS  - 0270-7306 (Linking)
VI  - 19
IP  - 10
DP  - 1999 Oct
TI  - Mutations altering the predicted secondary structure of a chloroplast 5' 
      untranslated region affect its physical and biochemical properties as well as its 
      ability to promote translation of reporter mRNAs both in the Chlamydomonas 
      reinhardtii chloroplast and in Escherichia coli.
PG  - 6980-90
AB  - Random mutations were generated in the sequence for the 5' untranslated region 
      (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding 
      sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, 
      and transformed into Escherichia coli, and white colonies were selected. Twelve 
      single base pair changes were found at different positions in the rps7 5'UTR in 
      207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation 
      of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA 
      expression cassette and transformed into the chloroplast of C. reinhardtii and 
      into E. coli. In vivo spectinomycin-resistant growth rates and in vitro 
      aminoglycoside adenyltransferase enzyme activity varied considerably between 
      different mutants but were remarkably similar for a given mutant expressed in the 
      Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on 
      aadA reporter expression and their complete abolition of lacZ' reporter 
      expression in E. coli suggests differences in the interaction between the 5'UTR 
      of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected 
      the predicted folding pattern of the 5'UTR by weakening the stability of stem 
      structures. Site-directed secondary mutations generated to restore these 
      structures in the second stem suppressed the loss of reporter activity caused by 
      the original mutations. Additional site-directed mutations that were predicted to 
      further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 
      leader both resulted in reduced reporter expression. This genetic evidence 
      combined with differences between mutant and wild-type UV melting profiles and 
      RNase T1 protection gel shifts further indicate that the predicted wild-type 
      folding pattern in the 5'UTR is likely to play an essential role in translation 
      initiation.
FAU - Fargo, D C
AU  - Fargo DC
AD  - Developmental, Cell and Molecular Biology Group, Departments of Botany and 
      Zoology, Duke University, Durham, North Carolina 27708, USA.
FAU - Boynton, J E
AU  - Boynton JE
FAU - Gillham, N W
AU  - Gillham NW
LA  - eng
GR  - GM-19427/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Mol Cell Biol
JT  - Molecular and cellular biology
JID - 8109087
RN  - 0 (5' Untranslated Regions)
RN  - 0 (RNA, Plant)
RN  - 0 (Ribosomal Proteins)
RN  - 0 (ribosomal protein S7)
RN  - EC 2.7.7.- (Nucleotidyltransferases)
RN  - EC 2.7.7.47 (streptomycin 3''-adenylyltransferase)
SB  - IM
MH  - 5' Untranslated Regions/chemistry/*genetics
MH  - Animals
MH  - Base Sequence
MH  - Chlamydomonas reinhardtii/*genetics
MH  - Chloroplasts/*genetics
MH  - Escherichia coli/genetics
MH  - Genes, Reporter
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Mutation
MH  - Nucleic Acid Conformation
MH  - Nucleotidyltransferases/biosynthesis
MH  - *Protein Biosynthesis
MH  - RNA, Plant/chemistry/genetics
MH  - Ribosomal Proteins/genetics
PMC - PMC84693
EDAT- 1999/09/22 00:00
MHDA- 1999/09/22 00:01
PMCR- 1999/10/01
CRDT- 1999/09/22 00:00
PHST- 1999/09/22 00:00 [pubmed]
PHST- 1999/09/22 00:01 [medline]
PHST- 1999/09/22 00:00 [entrez]
PHST- 1999/10/01 00:00 [pmc-release]
AID - 0758 [pii]
AID - 10.1128/MCB.19.10.6980 [doi]
PST - ppublish
SO  - Mol Cell Biol. 1999 Oct;19(10):6980-90. doi: 10.1128/MCB.19.10.6980.