Protein Biosynthesis Away from the Ribosome Genetic code Charging tRNA Ribosomes On the Ribosome...

Protein Biosynthesis

• Away from the RibosomeGenetic code

Charging tRNARibosomes

• On the RibosomeInitiation complex

Elongation factorsPeptide bond

formation Termination

Characteristics of the Code• non-overlapping

• degenerate

• triplet

• basically universal over all living species

• polar

• no punctuation

• subject to miscues

• defines a reading frame

OLD MAN AND THE SEAErnest Hemmingway

Insert AOLD MAA NAN DTH ESE A

OLD MAA ANA NDT HES EA

OLD MAA ANA AND THE SEA

OLD MAA NAD THE SEA

Insert A A

Insert AAA

Insert Adelete N

N

^

Framing the Code

Garbled

In frame

In frame

Garbled

tRNA

Charging tRNAQ: What is meant by ChargingA: Charging means placing an amino acid on the

3’ (acceptor) end of the tRNA

Q: So, what’s the big deal?

A: There are 20 amino acids; the code is degenerateThere could be 4 “isoaccepting tRNAs” competing for one

Q: I still don’t see a problem

A: One enzyme must recognize 4 different tRNA species and select the correct amino acid.

Q: One enzyme does all that?

A: No, each tRNA has its own enzyme

Q: What is this enzyme called?

A: Its call Aminoacyl-tRNA Synthetase

Q: So, there are 20 of these enzymes

A: Yes

Q: That makes the job a recognition a little easier then?

A: Yes, but the enzymes still have to distinguish between look-alikes such as leucine and valine, glutamine and glutamate, tyrosine and phenylalanine.

Q: Are all aminoacyl-tRNA synthetases alike?

A: Yes and no. Yes, they perform the same function,i.e., to recognize and transfer the correct amino acid to tRNA.Q: Why no?

A: Because one class (Class I) looks for the anticodon on the tRNA, the other (Class II) looks for other features.

Q: What else?

A: Class I puts the amino acid on the 2’ position of theterminal ribose on tRNA, Class II only the 3’.

Q: So, how does aminoacyl-tRNA synthetase discriminateamino acids and different tRNA species?

A: The key lies in the tRNA itself. Besides the anticodon,tRNAs have other bases that set them apart. These basescalled “identity elements” are found in the terminal ends(acceptor stem) and internal in the tRNA.

Q: Do they also proofreading?A: Yes, but sparingly

Q: How sparingly?

A: Enough to keep errors down to isoleucine mistaken for a valine once every 50,000 times. Ile-tRNA synthetaseactually hydrolyzes the valine-AMP precursor.

Reaction:

CH3

CH3

CH2-CH2CH-COO-

NH3+

+ ATP

CH3

CH3

CH2-CH2CH-CNH3

+

~O

O-P-O-CH2

O Ad

OHHO

O

O

CH3

CH3

CH2 CH-COO-

NH3+

L-Valine

Enzyme Bound

tRNA

L-Leucine

CH3

CH3CH2

CH-COO-

NH3+

CH2

L-Isoleucine

PPi

Codon-Anticodon Interactions

Polarity

5’ 3’3’ 5’

Codon on mRNA

Anticodon on tRNA

Anticodon loop

3’ 5’

C G I

(C, U)G C AmRNA 5’ 3’

Wobble base on anticodon

3rd position

mRNAs are always read 5’ to 3’.

mRNAs are always read 5’ to 3’.

Alanine

Ribonucleoprotein Particles

Ribosomes: The Staging Areas of Protein Synthesis

30S (40S)

16S RNA (18S)23 Peptides (33)

50S (60S)

70S (80S) Monosomes

23S RNA (28S)31 Peptides (49) 5S RNA (5S + 5.8S)

* Mammalian

50S30S

tRNA sites

mRNA

Crevice

Polysomes

Groups of ribosomes attached to a single mRNA