Review session directly following the talk. SL 110

Lecture Connections27 | 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 a great 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

Cricks’ Adaptor Hypothesis

• It was recognized in the1950s 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

Crick's adaptor hypothesis

Early History of Protein Synthesis Research

• 1955: Paul Zamenick’s group identifies ribosome as the site of protein synthesis

• 1956: Volkin and Astrachan find RNA that is like DNA but do not realize the importance of this finding

• 1958: Zamenick and Hoagland find that amino acids became attached to what is now known as tRNA

• 1960: Sydney Brenner realizes at a 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 acids

• Obviously a one letter code is too short (4)

• Four letter code in groups of two is insufficient (16)

• Four letter code in groups of three is sufficient (64)

• Living organisms use non-overlapping mRNA code

– One mRNA region encodes for one protein

Amino Acid Codons

• Written in the 5’ 3’ direction

• Third base is less important

Generally, only one reading frame codes for anything, BUT…

Translational frameshifting in a retroviral transcript. The gag-pol overlap region in Rous sarcoma virus RNA is shown.

Shifty nucleotidesShifty nucleotides

gag is one of the three "main" genes found in all retroviruses (along with env and pol). It contains around 1500 nucleotides, and encodes four separate proteins which form the building blocks for the viral core: Capsid protein, CA, p24, Matrix protein, MA, p17 (this protein isn't actually part of the viral core but the "matrix" which anchors the core to the viral envelope)Nucleocapsid protein, NC, p9 and p6.More about these later…

27-10 RNA editing of the transcript of the cytochrome oxidase subunit II gene from Trypanosoma brucei mitochondria. (a) Insertion of four U residues (pink) produces a revised reading frame. (b) A special class of guide RNAs, complementary to the edited product, act as templates for the editing process. Note the presence of two G=U base pairs, signified by a blue dot to indicate non-Watson-Crick pairing.

Deamination reactions that result in RNA editing. (a) The conversion of adenosine nucleotides to inosine nucleotides is catalyzed by ADAR enzymes. (b) Cytidine to uridine conversions are catalyzed by the APOBEC family of enzymes. (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like)

Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene.

RNA editing of the transcript of the gene for the apoB-100 component of LDL. Deamination, which occurs only in the intestine, converts a specific cytidine to uridine, changing a Gln codon to a stop codon and producing a truncated protein.

Nuclease cleavage

rRNA Operon7 copies/genome in E. coli

CCA Nucleotidyl transferase(template independent)

Splicing

Nuclease cleavage

A selection of the modified nucleosides that occur in tRNAs together with their standard abbreviations.

Three-dimensional structure of yeast tRNAPhe deduced from x-ray diffraction analysis. The shape resembles a twisted L.

CCA

Figure 32-8 Base sequence of yeast

tRNAAla drawn in the cloverleaf form.

Figure 32-9 Cloverleaf secondary structure of tRNA.

Figure 32-12 Tertiary base pairing interactions in yeast tRNAPhe.

Page

129

6

A selection of the modified nucleosides that occur in tRNAs together with their standard abbreviations.

Pag

e 12

94

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

Table 32-5 Allowed Wobble Pairing Combinations in the Third Codon–Anticodon

Position.

Pag

e 13

08

Wobble pairing. U · G and I · A wobble pairs. Both have been observed in X-ray structures.

Page

130

7

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

mRNA/tRNA binding is antiparallel

• 5’

5’ 3’mRNA

tRNA

CCA-amino acid (3’end)5’end

Codon: 1 2 3

Anticodon 3 2 1

Anticodon 3’---GUI----5’ Codon 5’----CCU----3’

Wobble base

Synthesis of Aminoacylated tRNAs: Aminoacyl AMP

Aminoacyl-tRNA Synthetases

• Each enzyme binds a specific amino acid and the matching tRNA

• Most cells contain twenty different aminoacyl-tRNA synthetases, one for each amino acid

• Some cells contain less than 20 synthetases; in this case one amino acid is converted to another after charging the tRNA

Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step 1 is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases

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 synthetase

• Only a few nucleotides in tRNA confer the binding specificity– Anticodon region

– Other regions (G•U in Ala-tRNA)

Gln-tRNA synthetase from E. coli, a typical monomeric class I synthetase

X-Ray structure of E. coli GlnRS · tRNAGln · ATP. tRNA and ATP wireframe; tRNA sugar–phosphates, bases, ATP.

Pag

e 13

00

PDB molecule of the month_the_month

Different Approaches to the Same ProblemIn this picture, five complexes of an aminoacyl-tRNA synthetase with tRNA are shown, aligned so that the tRNA molecules are in the same orientation. Notice that the enzymes approach the tRNA from different angles. The isoleucine, valine and glutamine enzymes cradle the tRNA, gripping the anticodon loop (at the bottom in each tRNA), and placing the amino-acid acceptor end of the tRNA in the active site (at the top right in each tRNA). These all share a similar protein framework, known as "Type I," approaching the tRNA similarly and adding the amino acid to the last 2' hydroxyl group in the tRNA.

The phenlyalanine and threonine enzymes are part of a second class of enzymes, known as "Type II." They approach the tRNA from the other side, and add the amino acid to the 3’ hydroxyl on the last tRNA base. Type I enzymes contain a Rossmann

fold in the catalytic domain (alternating αβ structures)

Rossmann fold from a decarboxylase

What’s bound to this enzyme????? Nicotinaminde mononucleotide

Figure 32-19a Comparison of the modes by which GlnRS and AspRS bind their cognate

tRNAs.

Page

130

2

(b) AspRS, a Class II synthetase.(a) GlnRS, a Class I synthetase.

Cartoon comparison of the putative aminoacylation and editing modes of IleRS ·

tRNAIle.

Page

130

4

http://pubs.acs.org/doi/full/10.1021/ja9095208

Structural elements of tRNAAla that are required for recognition by Ala-tRNA synthetase. (a) The tRNAAla structural elements recognized by the Ala-tRNA synthetase are unusually simple. A single G=U base pair (pink) is the only element needed for specific binding and aminoacylation. (b) A short synthetic RNA mini-helix, with the critical G=U base pair but lacking most of the remaining tRNA structure. This is aminoacylated specifically with alanine almost as efficiently as the complete tRNAAla.

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

Composition of E. coli Ribosome

• 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

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: