BCH 6740: Ribosomes

Homework: “Crystal Structure of the Ribosome at 5.5 Å Resolution”Marat M. Yusupov, Gulnara Zh. Yusupova, Albion Baucom, Kate Lieberman, Thomas N. Earnest, J. H. D. Cate, Harry F. Noller, Science, Vol. 292, Issue 5518, 883-896, May 4, 2001

“Structure of the bacterial ribosome at 3.5 Å Resolution”B.S. Schuwirth, M.A. Borovinskaya, C.W. Hau, W. Zhang, A. Vila-Sanjurjo, J.M. Holton, and J.H.D. Cate, Science, Vol. 310, 827-834, Nov. 4, 2005

Chain elongation in a prokaryotic systemEF ≡ Elongation Factor protein

aatRNA has free amino endPeptidyl chain is ester-linked to tRNA in

P siteEster link is replaced by amide bond to

amino acid on the tRNA in the A site by peptidyl transferase

∴ Protein synthesis is NH3+ COO-

“translocation”

Bacterial ribosomes: decomposition

2904 res.

120 res.

1542 res.

Bacterial ribosomes

50S+ 30S 70S complex

Monoribosome complex

KCl ↓↑ Mg2+

subunits

E. coli: 15,000 ribosomes(25% of dry mass)

50-120 msec/codon

Chemical cross-linking

unlinked

linked

Immuno-electron microscopy

Determining where a protein exits the ribosome

Antibodies are to β-galacto-sidase being synthesized

Antibody links two ribosomes in a particular orientation based on where the antigens are

Note: all three positions are NOT occupied at the same time

Triangles ≡ A siteCircles ≡ P siteSquares ≡ E site

Chemical cross-linking info:

Actual structures of the major players

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.

Ban N, Nissen P, Hansen J, Moore PB, Steitz TA.Science. 2000 Aug 11;289(5481):878-9.

tRNAMetf Nin the P site of the 70S ribosome at 5.5 Å

This is an example of the detail obtained at 5.5 Å resolution. You can see general shapes but are lacking a lot of atomic detail.

Entire complex

A) Looking at 30S

C) Looking at 50S

Parts

tRNAs

rRNA forms the scaffold

16S

23S

Subunits move relative to each other

Regions of large differencesbetween individual 50S or 30S and 70S structures

30S, 70S T. thermophilis50S H. marismortui

Proteins dress the outside of RNA

tRNA interactionsThis and other structures provide detail about the fidelity of translation

Anticodon stem loops bound to 30S

Other contacts with 50S

P-site interactions

A-site tRNA contacts

Detail is fuzzy at 5.5 Å

E-site tRNA

Kinked relative to tRNA crystal structure

The ribosome is a ribosyme

Large numbers of unique RNA folds

Many of the proteins have unusual or extended structures.

Subsequent work has tracked the mRNA in the ribosome

Yusupova, Yusupov, Cate, & Noller, Cell 106, 233-41 (2001)

This is how the ribosomes form in the crystal lattice. The red tubes are mRNA, and this suggests how they might form polysomes.

The E site of one ribosome is adjacent to the A site of another.

Eukaryotic ribosomes

18 sRNA, ~30 proteins5 sRNA, 5.8 sRNA, 28 sRNA, ~40 proteins

Protein translation on the endoplasmic reticulum