Lecture Connections 27 | Protein Metabolism

Protein Synthesis is a Very Complex Process• Occurs in a large macromolecular assembly called the ribosome, which is made of many proteins and several RNA molecules into a functional rib

Lecture Connections

27 | Protein Metabolism

© 2009 W H Freeman and Company

Protein Synthesis is a Very Complex Process

• Occurs in a large macromolecular assembly called the ribosome, which is made of many proteins and several RNA molecules into a functional ribosome

• Uses a large number of tRNAs that interact with mRNA

• Each tRNA is charged with one of the 20 common amino acids by 20 different aminoacyl tRNA synthetases

• Requires a dozen or more additional protein factors for initiation, elongation, and termination of translation

• Involves perhaps 100 additional enzymes that carry out a variety of modifications with many proteins

Protein Synthesis Occurs on

Ribosomes

• In eukaryotes, most ribosomes are attached to the cytosolic face of endoplasmic reticulum

Cricks’ Adaptor Hypothesis

• It was recognized in early 1950s that protein sequence is encoded by the nucleotide sequence

• The molecular nature of players in protein synthesis

remained unknown

• Crick proposed around 1954 the adaptor hypothesis

– template is RNA (maybe rRNA)

– the amino acid is carried to the template by an adaptor – adaptors may contain nucleotides

Early History of Protein Synthesis

• 1958: Zamenick and Hoagland find that amino acids

became attached to what is now known as tRNA

• 1960: Sydney Brenner realizes at Good Friday meeting with Crick and Francois Jacob and that the DNA-like

RNA of Volkin and Astrachan is the messenger RNA

The Genetic Code for Proteins Consists of Triplets of Nucleotides

• There are 20 common, genetically encoded

Amino Acid Codons

• Written in the 5’  3’ direction

• Third base is less important

Most Amino Acids Have More Than

One Codon

• Only Met and Trp have a single codon

• Some codons are better than others because of abundance of such tRNAs

Genetic Code is Nearly Universal

Molecular Recognition of Codons in

mRNA by tRNA

• The codon sequence is complementary with the anticodon sequence

• The codon in mRNA base pairs with the

anticodon in mRNA via hydrogen bonding

• The alignment of two RNA segments is

antiparallel

Inosinate in the Anticodon in

Some tRNAs is “Wobble”

• Inosinate can hydrogen bond with three

different nucleotides (A, U, C)

• This interaction is weaker than typical Watson Crick base pairing

Some mRNAs are Edited Before

Protein Synthesis

• Editing involves alteration, addition, or deletion

of nucleotides in mRNA

• Editing uses guide RNA that temporarily

hybridizes with the mRNA and acts as a

template for editing

Protein Synthesis Involves Five

Stages

• Activation of amino acids

– Enzymatic synthesis of aminoacyl tRNA molecules

• Initiation of translation

– Binding of mRNA and N-formylmethionine to ribosome

• Elongation

– Binding of aminoacyl tRNAs to ribosome

– Formation of peptide bonds

• Termination and ribosome recycling

– Termination codon in mRNA reaches ribosome

• Folding and post-translational processing

– Catalyzed by a variety of enzymes

Key Players in Protein Synthesis

• Ribosome is a Non-covalent Assembly of Many Proteins and Few RNA Molecules

30S and 50S Subunits of Bacterial

Ribosome

• Subunits are identified by their sedimentation

coefficients (Svedberg units)

• A, P, and E sites are locations for binding of

tRNA molecules

The Assembled Bacterial Ribosome

Ribosomal RNA Molecules have Complex Secondary Structures

Transfer RNA Molecules have a Characteristic Secondary Structure

Transfer RNA Molecules Fold into Twisted L-Shaped Structure

Synthesis of Aminoacylated tRNAs:

Aminoacyl AMP

Synthesis of Aminoacylated tRNAs:

Transfer of Aminoacyl to tRNA

aminoacyl-• Some cells contain less than 20 synthetases;

in this case one amino acid is converted to

another after charging the tRNA

The Second Genetic Code

• Matching each amino acid with correct tRNA can be viewed as the “second genetic code”

• The “code” is in molecular recognition of a specific tRNA molecule by a specific

22 Genetically Coded Amino Acids

• 20 genetically encoded amino acids are

common in all organisms

• Selenocysteine is formed after charging an

UGA(stop)-recognizing tRNA with serine in both bacteria and eukaryotes

• Pyrrolysine is directly attached to its tRNA that recognizes UAG(stop) codon by some archae

Formation of the Initiation Complex

• The mRNA is guided to the 30S subunit by

Shine-Dalgarno sequence in mRNA that is

complementary to a sequence in ribosomal

RNA

• Formylmethionine tRNA bids to the peptidyl

site

• Large 50S subunit combines with the 30S

subunit forming the initiation complex

Several Protein Factors are

Involved in Initiation

First Elongation Step

• Aminoacyl tRNA binds to the elongation

factor Tu that also carries GTP

• The EF-Tu-GTP complex with second amino acid on its tRNA binds to the aminoacyl site

• After GTP hydrolysis EF-Tu-GDP leaves the ribosome

Formation of the Peptide Bond

• The 23S RNA ribozyme is the catalyst

Termination of Protein Synthesis

• When the stop codon (UAG) reaches

ribosome a release factor binds to the A site

• The linkage between nascent polypeptide and tRNA in the P site is cleaved

• Protein, mRNA, and deacylatd tRNA

dissociate from ribosome

Coupling of Transcription and

Translation in Bacteria

Chapter 27: Summary

• The primary genetic code for protein synthesis is in triplets of nucleotides in mRNA that recognize the anticodon of tRNA

• The secondary genetic code for protein synthesis is in

recognition features between tRNA and the enzyme that

attaches the correct amino acids to this tRNA

• The mRNA binds to the ribosome and its codons are

exposed to the aminoacyl-tRNA binding site

• The protein synthesis from charged aminoacyl tRNA

substrates is catalyzed by a ribozyme in the ribosome

In this chapter, we learned that: