Differential regulation of plastid mRNA stability. Progress report

Technical Report · Wed Sep 01 04:00:00 EDT 1993

DOI:https://doi.org/10.2172/10175480· OSTI ID:10175480

Stern, D B

Our goal is to identify cis-acting sequences and transacting factors that function in plastid mRNA maturation, stabilization, and/or decay through an in vitro and in vivo analysis of mRNA:protein interactions. Our previous results emphasized the study of 3{prime}end inverted repeat sequences (IRs) that serve both as mRNA processing elements and stability determinants, and associate with plastid proteins that potentially play enzymatic, structural and/or regulatory roles. We seek to define, by single base and internal deletion mutagenesis, the sequence and structural requirements for protein binding to the 3{prime} IRs of petD and psbA mRNAs; to purify RNA-binding proteins that demonstrate gene- or sequence-specific binding, or that are implicated in RNA stabilization or decay; and to investigate the native form of mRNA in the plastid, by attempting to purify ribonucleoprotein (RNP) particles from organelles. Our view of mRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNA-binding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with petD 3{prime} IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the petD stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins.

• Research Organization: Boyce Thompson Inst. for Plant Research, Ithaca, NY (United States). Environmental Biology Dept.
• Sponsoring Organization: USDOE, Washington, DC (United States)
• DOE Contract Number: FG02-90ER20015
• OSTI ID: 10175480
• Report Number(s): DOE/ER/20015--2; ON: DE93019889
• Country of Publication: United States
• Language: English

Related Subjects

550201
59 BASIC BIOLOGICAL SCIENCES
AUTORADIOGRAPHY
CHLOROPLASTS
DNA SEQUENCING
ELECTROPHORESIS
FRACTIONATION
GENE MUTATIONS
MOLECULAR BIOLOGY
NUCLEASES
NUCLEOPROTEINS
PHOSPHORUS 32
PROGRESS REPORT
RNA
RNA PROCESSING
STRUCTURE-ACTIVITY RELATIONSHIPS
TRACER TECHNIQUES