Chapter 33. Biochemistry 432/832 Protein Synthesis and Degradation December 05. Announcements. Central dogma of biochemistry: DNA --> RNA --> protein. Transcription Translation. Protein Synthesis and Degradation. Outline. 33.1 Ribosome Structure and Assembly
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Protein Synthesis and Degradation
DNA --> RNA --> protein
Protein Synthesis and Degradation
Proof that polypeptides (hemoglobin) grow by addition of new amino acid residues to the carboxyl end
Direction of chain growth
Dintzis experiment, 1961, rabbit reticulocytes labeled with leucine
Ribosomal RNA operons in Escherichia coli. Precursor RNA is cleaved to generate 23S, 16S and 5S rRNA as well as tRNA
The rest of space (peripheral) is occupied by proteins
Image reconstruction is based on cryoelectron microscopy
Two binding sites
Transfer of a polypeptide to the amino group of amino acid carried by the tRNA in the A site
Aminoacyl tRNA is at the A site; polypeptidyl-tRNA is at the P site
One codon translocation; tRNA expulsion
Differences with other tRNAs
30S subunit (IF-3:IF-1) binds mRNA
IF-2 delivers the initiator f-Met-tRNA to the P site
IF-2 dissociates from 30S subunit
GTP hydrolysis is accompanied by IFs release and binding of the 50S subunit
Allowed separation of GTP binding from GTP hydrolysis
Factor Mass Molecules/Cell Function
Reaction of the tRNA-linked peptidyl chain with the a-amino group of an adjacent aminoacyl-tRNA -
no energy is required for activation
Catalytic center is located in the 50S particle
The peptidyl transferase center of 23S rRNA
Puglisi JD, Blanchard SC, Green R, Nat Struct Biol 2000 Oct;7(10):855 Approaching translation at atomic resolution.
Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction
In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme
In the 30S structure, tRNA binding sites were located
The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis
Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF.
Science 2001 Mar 29
Crystal Structure of the Ribosome at 5.5 A Resolution.
We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound mRNA and tRNAs at 5.5 A resolution. All of the 16S, 23S and 5S rRNA chains, the A-, P- and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 A movements associated with tRNA translocation with intersubunit movement.
Relative positions of tRNA molecules in a ribosome during peptidyl transfer and translocation