Translation

Lecture 7 of

Introduction to Molecular Biology

生理所 蔡少正

The Central Dogma

The information in nucleic acid can be perpetuated or transfer (from DNA to RNA to Protein), but the transfer of information to protein is irreversible.

A(A)nA

Translation: the process of translating the sequence of nucleotide bases in DNA/RNA into a sequence of amino acids in a protein

Genetic Code

The genetic code: Collection of base-sequences (codons) that corresponds to each amino acid and to translation signals.

DNA RNA Protein

A, T, G, C A, U, G, C 20 amino acids

-The genetic code contains three bases-Most amino acids (except methionine and tryptophan)

have more than one codon.-The triplet code is univeral with a few exceptions. e.g.

mitochondria, certain yeast, chloroplast use different codes

Universal Codon

Reading FrameA reading frame refers to one of three possible ways of reading

a nucleotide sequence.

E.g. acttagccgggacta

reading frame:

123

|||

acttacccgggacta

1st T Y P G L

2nd L T R D

3rd L P G T

Transfer RNATransfer RNA (tRNA) as an

adapter between base sequence and amino acid sequence: The tRNA molecules are small, single-stranded nucleic acids ranging in size from 73 to 93 nucleotides. Due to pairing of complementary base sequences, the tertiary structure of a tRNA is L-shaped.

tRNAThree regions of each tRNA

molecule are used in the decoding operation:

1. Anticodon region: form base pairing with mRNA

2. Amino acid attachment site: specific amino acid covalently linked to this region

3. Recognition region:As yet ill defined multiple parts, for tRNA distinction

Aminoacyl-tRNA SynthetaseThere is at least one tRNA (usually more) for

each amino acid. The amino acid is covalently linked to corresponding tRNA by aminoacyl-tRNA synthetase. The different tRNA molecules and synthetases are designated by stating the name of the amino acid that can be linked to a particular tRNA. For example, leucyl-tRNA synthetase attaches leucine to tRNAleu. When an amino acid is attached to a tRNA molecule, the tRNA is said to be acylated or charged.

Wobble HypothesisCodon (mRNA)-anticodon (tRNA) interaction and

Wobble hypothesis: The identity of the third codon-base appears to be unimportant. But two steric constraints exists.

(a) Two purines cannot pair with one another because there is not enough space for a planar purine-purine pair.

(b) Two pyrimidines cannot pair because they cannot reach one another.

GCU GCC GCA Alanine

tRNA anticodonCGI CGI CGI

Ribosome-A multicomponent particle containing several

enzymes needed for protein synthesis.

-Locates in cytoplasm

Prokaryote (70S) Eukaryote (80S)30S 50S 40S 60S1 16S rRNA, 1 23S rRNA, 18S rRNA 1 5S, 1 5.8S,

1 5S rRNA 1 28S21 proteins 32 proteins 30 proteins 50 proteins

-Each ribosome contains two cavities into which tRNA molecules can be inserted: the P (peptidyl) site and the A (aminoacyl) site.

InitiationMany factors are involved and AUG is always

used as the initiator.

Only tRNAiMet (tRNA carries the very first amino

acid) can directly enter into P site, all other aminoacyl-tRNAs enter A site.

Model for Eukaryotic Initiation

ElongationThe polypeptide chain is elongated by transferring the

polypeptide attached to tRNA in the P site to the aminoacyl-tRNA present in the A site (catalyzed by peptidyl transferase). The newly synthesized (n+1) polypeptide is then translocated to the P site so the A site is vacant for next aminoacyl-tRNA complex.

Elongation

Termination

There are 61 codons assigned to amino acids, the other 3 triplets (UAA, UAG, and UGA) are termination (stop) codons. No tRNA reprenents any of these three stop codons (That's why they are stop codons!). The stop codons are recognized by release factors (RFs), which help release of the completed polypeptide from the last tRNA. Termination reaction involves expulsion of the tRNA from the ribosome, and dissociateion of the ribosome from mRNA.

Polycistronic vs. Monocistronic

mRNA 1 2 3AUG Stop AUG Stop AUG Stop

Using Prokaryotic ribosomes

Using eukaryote ribosomes

Prokaryotic ribosomes can read-through the stop codon and re-initiate initiation complex for the second gene while eukaryotic ribosomes fall of at the stop codon

The accuracy of translation

There are two stages in protein synthesis at which errors might be made:

(a) Charging a tRNA only with its correct amino acid: less than 1 in 105 aminoacylation. If all possible amino acid misacylations occur at this rate, only about 0.17% of the proteins would be defective.

(b) Recognition of codon-anticodon: 1 in 10 to 1 in 100.

Kinetic Proofreading

The second stage seems like a weak point and is not the case for protein synthesis, so there must be some kind of proofreading during translation process. This is called kinetic proofreading: Mismatched aminoacyl-tRNA dissociates more rapidly than correctly matched aminoacyl-tRNA (~500X); therefore, increasing time spent in the A site before peptide bond formation occurs increases the probability that the correct aminoacyl-tRNA will be utilized.

The overall error rate in protein synthesis is ~ 5 X 10 -4 per codon

Coupled Transcription-TranslationPolyribosome (polysome): more than one ribosomes were seen in the same mRNA moleculeIn prokaryote, translation usually starts while transcription is still ongoing

Consisting of more than one peptide

Folding of -helix and -sheets

Amino acids are linked by H-bonds

Sequence of amino acids

Protein Structures