Chapter 19

Ribosomes and Transfer RNA

Ribosome composition• Prokaryotes

– 30S• S1-S21 + 16S rRNA

– 50S• L1-L33 (L34 not visible) + 5S rRNA and 23S rRNA

• Eukaryotes– 40S

• About 30 proteins + 18S rRNA

– 60S• About 40 proteins + 5S, 5.8S, and 28S rRNA

Fig. 19.5

Polysomes -P625

Both prokaryotes and eukaryotes have polysomes

Polysomes -P624

Most mRNA are translated by more than one ribosome at a time; the result, a structure in which many ribosomes translate an mRNA in tandem, is called a polysome. In eukaryotes, polysomes are found in the cytoplasm. In prokaryotes, transcription of a gene and translation of the resulting mRNA occur simultaneously. Therefore, many polysomes are found associated with an active gene.

74 ribosome translatingsimultaneously

Eukaryotes

Prokaryotes

19.2 Transfer RNA

The discovery of tRNA

• Zamecnik and et al.,1957

• pH5 enzyme fraction works with ribosomes to direct translation of added mRNAs.

Mix RNA with pH5 enzyme, ATP and [14C]Leucine

Charging of tRNA with an amino acid

Mix [14C]Leucine-charged pH 5 RNA with microsome

Incorporation of leucine from leucyl-tRNA in to the nascent protein on ribosome

Summary

• Transfer RNA was discovered as a small RNA species independent of ribosomes that could be charged with an amino acid and could then pass the amino acid to a growing polypeptide.

tRNA structure• Holley and et al, 1965 first determined the alanine tRNA

structure from yeast • The common features of tRNA (from at least 14 tRNAs)

– Acceptor stem• Including the two ends of the tRNA and invariant sequence

CCA at 3’ end

– D (dihydrouracil) loop– Anticodon loop-all important– Variable loop

• Contain 4 to 13 nt

– T loop• TψC: ψ stands for pseudo-uridine

The second genetic code

Recognition of tRNAs by aminoacyl-tRNA synthetase:

AMP/amino acid coupling

AMP/tRNA displacement

Ribosome recognize the tRNA but not the amino acid

• Lipmann, Benzer, von Ehrenstein and et al, 1962

• Cysteinyl-tRNAcys →Alanyl-tRNAcys

• In vitro translation using poly(UGU) as template—this does not contain any codon for alanine

• Codon for ala is GCN, cysteine is UGU• Alanine is incorporated instead of cysteine• Fig. 19.28

It is the nature of the tRNA that matters

Fig. 19.28

Given that the secondary and tertiary structures of all tRNA are essentially the same, what base sequences in tRNA do the synthetases recognize when they are selecting one tRNA out of a pool of over 20?

• The acceptor stem ?

• The anticodon ?

Summary

• Biochemical and genetic experiments have shown that the anticodon, like the acceptor stem, is an important element in charging specificity.

• Sometimes the anticodon can be absolute determinant of specificity.

Proofreading and editing by aminoacyl-tRNA synthetases

1958, Pauling used thermodynamics and found:

Ile and val only differ in –CH2 group and one-fifth Val-tRNAile would be made

In fact, 1/150 amino acid is activated by IleRS to make Val and 1/3,000 aminoacyl-tRNA is Val-tRNAile

How does isoleucyl-tRNA synthetase prevent formation of Val-tRNAIle?

Double-sieve mechanismFersht in 1977

How does isoleucyl-tRNA synthetase prevent formation of Val-tRNAIle?

Fig. 19.31

Activation site

Fig. 19.32

The space between Trp232 and Tyr386 is just big enough to accommodate valine but not Ile (too big)Abolish this region could abolish editing but not activation activity

Fig. 19.33

Summary-1• The amino acid selectivity of at least some aminoacy

l-tRNA synthetases is controlled by a double sieve mechanism.

• The first sieve is a coarse one that excludes amino acids that are too big. The enzyme accomplishes this task with an active site for activation of amino acids that is just big enough to accommodate the cognate amino acid, but not larger amino acids.

• The second sieve is a fine one that degrades aminoacyl-AMPs that are too small.

Summary-2• The enzyme accomplishes this task with a second ac

tive site (the editing site) that admit small aminoacyl-AMPs and hydrolyzes them. The cognate aminoacyl-AMP is too big to fit into the editing site, so it escapes being hydrolyzed. Instead, the enzyme transfers the activated amino acids to its cognate tRNA