Journey of a protein molecule

I: A protein is born

1. Translation1) What is translation?

2) Where is translation occurs in plant cells? Three genomes!

3) What do you need for translation?The blueprint:The building blocks:The carrier/porters:The catalyzers:The factory:

4) What is the process?

Initiation—elongation---termination

5) Regulation of the trnaslation

a) Initiation establishes the reading frame of the mRNA and position the first amino acid for incorporation

Step 1 Major reactions include connection of amino acid with its tRNA (enzyme called aminoacyl-tRNA synthetase), Met-tRNA interaction with 40S small ribosome subunit, and cap interaction of initiation factors IF4.

Step 2 involves interaction of 40S ribosome and tRNA-AA complex with mRNA near the cap structure with the help of IF4

Step 3 involves scanning of the mRNA by the 40S-tRNA-AA complex until finding the first codon AUG (the anitcodon on the tRNA is UAC)

Step 4: release of the IFs and formation of the intact ribosome-mRNA cmplex. The Met residue is placed into the P site.

Points of complexity:

*IFs consist of many members that are highly conserved in eukaryotes including plants. Six major classes are identified (IF1—IF6). Different class has distinct function in the initiation process. For example, IF2 mediates GTP-dependent recognition of Met-tRNA; IF5 promote the GTPase activity of IF2 thereby release of IFs from the ribosome.

**The speed of initiation is highly regulated by many factors. For example, temperature fluctuation, hormones, light, pathogen attack etc can all modulate the speed of specific protein synthesis. One recently discovered mechanism is through activity of IF2---a guanine nucleotide exchange factor called IF2B mediates GDP release from IF2 so that IF2 can bind GTP again to function. Phosphorylation can be important for IF2 activity as well.

b) Elongation: is sequential addition of amino acids to the existing protein chain. A major reaction is the formation of peptide bond between the adjacent AAs. This is done by an enzyme called peptidyl transferase—a ribozyme in the large subunit of ribosome.Three sites on the ribosome—P, A, E.Elongation factors (EFs) are important regulators that interact with tRNA-AA complex and releases after finding the A site.Proofreading: how could cell make sure the AA added to the chain is correct?

c) Termination: termination factor recognize the stop codon (UAG, UGA, UAA) and allow the peptidyl transferase to cleave the tRNA from the last AA.

2. Protein synthesis in chloroplasts

1) importance: 50-100 proteins insides the chloroplast are encoded by plastid genome

2) Distinct features of chloroplast protein synthesis as compared to cytoplasmic process:

a) The mRNA is not cappedb) the mRNA is often polycistronic (more than one proteins

are encoded by the same mRNA)c) Secondary structure of mRNA plays critical role for

control of initiation rate.d) 30S ribosome involved in selection of the starting codon

through a rRNA that can pair with a mRNA sequence before the AUG.

e) The initiation factors are different.f) Other mRNA binding proteins play a role in regulationg) The systems are not interchangable.

3) Regulationa) Stromal and thylakoid membrane can both be site of

translation. The thylakoid membrane targeting may be done by the nascent chain at he N-terminal. Details need to work out.

b) Light regulation of translation:

light controls the transcription, mRNA stability, but also the translation rate.

c) How does photosynthesis connect to protein synthesis in the chloroplast?

Redox regulation: mRNA binding proteins at the 5’ UTR play a significant role in mRNA translation rate. The photosynthetic electron transport chain activity produces ATP/NADPH—reducing power used to reduce many proteins. One of such proteins is a mRNA binding protein. After reduction, this protein would jump on the mRNA encoding the photosynthetic protein and produce the protein at much faster pace---as positive feedback from light---photosyntheisis light reaction---more protein synthesis.

ATP/ADP ratio: This ratio drops in the dark, increase in the light. More ADP will phosphorylate the PDI that cannot reduce mRNA binding factor and turn off the translation.

d) Availability of cofactors controls the translation

Many photosynthetic proteins like cytochromes and other electron carriers need metal to assemble into functional protein. If there is no such cofactor available, the protein synthesized will not be “useful”. So cells develop mechanism to coordinate translation of the protein ---if there is no cofactor, no synthesis.