TranslationProtein biosynthesis

Review of Steps in Gene Expression

RNA = nucleotide sequence

protein = amino acid sequence

Adaptor molecule

Translating the Message

How does the sequence of mRNA translate into the sequence of a protein?

• What is the genetic code? • How do you translate the "four-letter code" of

mRNA into the "20-letter code" of proteins? • And what are the mechanics like? There is no

obvious chemical affinity between the purine and pyrimidine bases and the amino acids that make protein.

• Three major advances gave the clues to solving this dilemma

Clue #1The Discovery that Proteins are made on

ribosomes

• By Paul Zamecnik (early 1950s) • He asked where in the cell are proteins

synthesized? • Injected rats with radioactive amino acids • A short time after injection (when the amino acids

should be incorporated into newly-synthesized proteins) he killed the rats, harvested their livers, ground them up and divided the cell components into “subcellular fractions” by centrifugation

Results• Radioactivity was found in small ribonucleoprotein

particles visible by electron microscopy. • These were later characterized and called

“ribosomes” (since they had RNA as a major component)

From Lehninger “Principles of Biochemistry” p 1021

Clue #2The Discovery that amino acids are

“Activated”

• By Hoagland and Zamecnik • They incubated amino acids with the cytosolic fraction

of liver cells, and with ATP • They found the amino acids became “activated”

during the incubation • Activation consists of attaching the amino acids to a

tRNA • Activated amino acids are called aminoacyl- tRNAs • The enzymes that do the activation are called

aminoacyl-tRNA synthetases

Clue #3Crick’s Adaptor Hypothesis

• Francis Crick thought about the problem

• He reasoned that a small nucleic acid could serve as an adaptor between RNA and protein synthesis if it could bind both RNA and an amino acid

• His idea was that one end of the adaptor would bind a specific amino acid and the other would bind to a specific sequence in the RNA that coded for that amino acid

•

Crick’s Adaptor Hypothesis

From Lehninger “Principles of Biochemistry” p 1021

•These adaptors are the tRNAs

• each tRNA can recognize specific sequences in the RNA transcript

•Each is “charged” with the amino acid that is specified by that sequence

What is the nature of the Code

• 1:1 correspondence can’t work • Therefore nucleotides must be read in combinations • Is 2 enough? 4X4 = 16 different combinations possible

- not enough • But 3 would give 4X4X4 = 64 combinations • This would be enough to code for 20 amino acids • Therefore the concept of the triplet codon was born

mRNA (nucleotides)

protein (amino acids)

4 different nucleotides

20 different amino acids

The Nature of the Genetic Code

• A group of three bases codes for one amino acid

• The code is not overlapping • The base sequence is read from a fixed

starting point, with no punctuation • The code is degenerate (in most cases,

each amino acid can be designated by any of several triplets)

Features of the Genetic Code • All the codons have meaning: 61 specify amino

acids, and the other 3 are "nonsense" or "stop" codons

• The code is degenerate - except for Trp and Met, each amino acid is coded by two or more codons

• Codons representing the same or similar amino acids are similar in sequence C Third-Base Degeneracy C 2nd base pyrimidine: usually nonpolar amino acid C 2nd base purine: usually polar or charged aa

Third-Base Degeneracy and The Wobble Hypothesis

• The first two bases of the codon make normal H-bond pairs with the 2nd and 3rd bases of the anticodon

• At the remaining position, non-canonical pairing may occur

• The rules:

Anticodon (base #1)

C A G U I

Codon (base #3)

G U

C,U A,G

U,C,A

tRNA• tRNAs are the “adaptors” in protein synthesis

Review of tRNA Structure

• There are many different tRNAs, each has a distinct sequence

• However, all tRNA have several conserved features

•

1) small (73-93 nucleotides long)

2) they have a conserved secondary structure - 4 stems and 4 loops with important functions

3) they contain many unusual bases

Inosine (I), pseudouridine (ψ ), dihydrouridine (D), ribothymidine (T), and methylated bases (mG, mI)

Invariant bases

Amino acid addition site

Varies in size

Interacts with the ribosome

Base pairs with the codon in the mRNA transcript

tRNA tertiary structure: L-shape

tRNAs are bifunctional

AAA Anticodon loopUUU

Codon in mRNA

Acceptor stemPhe

specific amino acid•Amino acids must be activated for translation

•Via covalent linkage of an amino acid to the 3’OH of the tRNA

•This generates a “charged tRNA”, aminoacyl-tRNA

•

tRNA activation must be specific•The delivery of the amino acid is specified by this codon-anticodon interaction (regardless of which amino acid is attached to the tRNA) • •Each tRNA is matched with its amino acid long before it reaches the ribosome. • •The match is made by a collection of remarkable enzymes, the aminoacyl-tRNA synthetases. • •These enzymes charge each tRNA with the proper amino acid, thus allowing each tRNA to make the proper translation from the genetic code of DNA into the amino acid code of proteins.

The Aminoacyl-tRNA Synthetase Reaction

• The goal of this reaction is to activate an amino

acid by forming an ester linkage with the correct tRNA

CC O-P-OCH2

O

O

O

-OHOH

Adenine

R group- -

H

NH3+

C

O C

CCAOH 3’ acceptor stem

Amino acid

The Aminoacyl-tRNA Synthetase Reaction is two steps

1) Activate the amino acid first, by reacting with ATP

2) Transfer the activated amino acid to its cognate tRNA

Amino acid + ATP Aminoacyl AMP + PPi

An enzyme-bound intermediate

2Pi

Aminoacyl AMP +tRNA Aminoacyl-tRNA + AMP

Aminoacyl-tRNA Synthetases

• All have a common 2-domain structure

A catalytic domain

Interacts with the tRNA 3’OH

Recognizes and binds the cognate amino acid

A variable domain

Interacts with the specific bases on the tRNA that identify that tRNA

Aminoacyl-tRNA Synthetases High fidelity in translation

• Aminoacyl-tRNA synthetases must perform their tasks with high accuracy, since every mistake will result in a misplaced amino acid when new proteins are constructed.

• These enzymes make about one mistake in 10,000.

• This is a two different levels:

1) They must be able to recognize and bind to the correct tRNA

2) They must be able to recognize and bind to the correct amino acid