BIO SCI 98 Lecture Notes - Lecture 5: Harry F. Noller, 23S Ribosomal Rna, Protein Folding
19 Jun 2018
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Translation overview
1) AA-tRNA
a) Activation of amino acids: the tRNA is aminoacylated
2) Initiation
a) The mRNA and the aminoacylated tRNA bind to the small ribosomal subunit
b) The large subunit binds as well
3) Elongation = translation
a) Successive cycles of aminoacyl-tRNA binding and peptide bond formation occur
until the ribosome reaches stop codon
4) Termination
a) Translation stops when a stop codon is encountered
b) The mRNA and protein dissociate and ribosomal subunits are recycled
5) Protein folding
Ribosomes are huge
- Each E. coli cell contains 15K ribosomes = 25% of
cell's dry weight
- Cells at peak proliferation capacity (24 min
doubling time) contain 72K ribosomes (⅓ mass of
cell)
- Bacterial ribosome
- ~7500 amino acids (57 subunits)
- ~4600 nucleotides (3 rRNAs)
- 20-30 nm across
- 2.5 MDa
- Bacterial RNA polymerase
- ~3500 amino acids (5 subunits)
- 5 nm across
- 400 kDa
- Nucleotides are bigger than amino acids
- Amino acid ~ 100 Da
- RNA nucleotide ~ 300 Da
Ribosomes contain 2 subunits
Bacteria
-Small subunit - 30s
- rRNA - 16s
- Protein - 21 total subunits (S1- S21)
- Function- mRNA, tRNA assembly
-Large subunit - 50s
- rRNA - 5S and 23S
- Protein - 36 total subunits (L1 to L36)
- Function - catalyze peptide bond
formation
Eukaryotes
- Similar to bacteria, but more complex and
more proteins
- 40s small and 60s large
find more resources at oneclass.com
find more resources at oneclass.com
rRNA processing
- rRNA transcribed as one long transcript (30S) then later processed into 16S, 23S, 5S
mature RNAs (note that these do not add up to 30)
-Svedberg units (S) - roughly proportional to size, but not linear relationship, based on
sedimentation rates during ultracentrifugation
Ribosome experiment
-Harry Noller
- Ribosome catalytic activity → what is responsible for peptide bond formation?
rRNA, protein, or both?
- In vitro translation assay
-Puromycin - mimics tRNA, but binds directly to the large subunit, ribosomes will
attach it to growing polypeptide chain
- New amino acids cannot be attached to puromycin, so it terminates
translation and is therefore a powerful antibiotic
- In this experiment, puromycin is just the substrate for forming peptide
bonds
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Translation overview: aa-trna, activation of amino acids: the trna is aminoacylated. Each e. coli cell contains 15k ribosomes = 25% cell"s dry weight. Cells at peak proliferation capacity (24 min doubling time) contain 72k ribosomes ( mass cell) Rna nucleotide ~ 300 da of of. Protein - 21 total subunits (s1- s21) Large subunit - 50s rrna - 5s and 23s. Protein - 36 total subunits (l1 to l36) Similar to bacteria, but more complex and. 40s small and 60s large rrna processing rrna transcribed as one long transcript (30s) then later processed into 16s, 23s, 5s mature rnas (note that these do not add up to 30) Svedberg units (s) - roughly proportional to size, but not linear relationship, based on sedimentation rates during ultracentrifugation. Puromycin - mimics trna, but binds directly to the large subunit, ribosomes will attach it to growing polypeptide chain.
