REGULATION OF BILIPROTEIN BIOSYNTHESIS IN CYANOBACTERIA

Project: Research project

Description

The long-term objectives of this research program are to develop a
detailed understanding of the structure, function, biogenesis, and
regulation of the cyanobacterial photosynthetic apparatus. The dual goals
of providing a detailed understanding of how light-harvesting anennae
function as well as providing molecular tools which are especially
important in characterizing gene regulation responses of cyanobacteria to
nutrients and light intensity are being pursued. The RNA polymerase of
Synechococcus sp. PCC 7002 apparently employs several different sigma
factors. The hypothesis that global patterns of gene expression in
cyanobacteria are controlled by promoter selection by these multiple sigma
factors will be tested. Sigma factor proteins will be overproduced in E.
coli and reconstituted with core RNA polymerase for in vitro transcription
studies. The long-term aim is to correlate sigma factors with specific
promoter sequences and with patterns of gene expression. All oxygen-
evolving photosynthetic organisms exhibit an adaptive phenomenon called
"state transitions" by which light energy is distributed between the two
photosystems to maximize the overall rate of photosynthetic election
transport. The ApcD gene product is required for state transitions to
occur and for energy to be transferred to Photosystem I. The hypothesis
that post-translational modification of ApcD controls light energy
distribution within the phycobilisome will be tested. Additional
experiments will seek to demonstrate the contact partner of ApcD on the
Photosystem I reaction center. Strains, plasmids, and methods to create
site-specifically altered phycocyanins have been generated. These tools
will now be used to address specific aspects of structure and function for
phycocyanin through site-directed mutagenesis. Spectroscopic analyses of
the mutant phycocyanins will be performed, and proteins exhibiting
interesting defects in light-energy harvesting will be studied in
collaboration with Dr. Robert Huber by X-ray crystallography. The ApcE
protein plays critical roles in energy transfer to Photosystem II as well
as in directing core assembly in the phycobilisome. Additional structure
and function studies will be performed on this interesting protein, and
the hypothesis that phycobilisome core structure is intrinsically
determined by the number of linker domains in ApcE will be tested. The
proposal that phycobilisomes of Anabaena sp. PCC 7120 contain four classes
of peripheral rods and a total of eight rods attached to their cores will
also be tested. The location of ferredoxin NADP+ oxidoreductase in
phycobilisomes will be studied by immuno-electron microscopy, and the role
of this molecule in phycobilisomes will studied by construction of a
mutant lacking the recently identified CpcD-like domain. Finally, a bank
of interposon- or transposon-tagged mutants will be created which will be
screened for mutations affecting phycocyanobilin synthesis, covalent
attachment of phycocyanobilin to phycobiliproteins, and state transitions.
The successful completion of this program should significantly extend
knowledge of mechanisms regulating gene expression in the photoautotrophic
cyanobacteria and will additionally provide significant new insights into
how light energy is efficiently harvested and distributed to the
photosystems.
StatusFinished
Effective start/end date3/1/832/29/04

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $265,646.00
  • National Institutes of Health: $144,404.00
  • National Institutes of Health
  • National Institutes of Health: $259,681.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $214,387.00
  • National Institutes of Health: $271,997.00
  • National Institutes of Health: $158,549.00
  • National Institutes of Health
  • National Institutes of Health: $73,100.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $212,133.00
  • National Institutes of Health

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Cyanobacteria
sigma factors
phycobilisome
Phycobiliproteins
Phycobilisomes
biosynthesis
Genes
energy
Phycocyanin
Synechococcus
Gene Expression
DNA-directed RNA polymerase
Lac Operon
Recombinant DNA
mutants
gene expression
transcription (genetics)
temperature
Gene Expression Regulation
Synechococcus sp. PCC 7002