reginald h. garrett charles m. grisham chapter 30 protein synthesis

Reginald H. GarrettCharles M. Grisham

Chapter 30Protein Synthesis

Outline

• The genetic code.• Matching an amino acid with its proper tRNA.• Condon-anticodon pairing.• Structure and assembly of ribosomes.• Events in mRNA translation.• Proteins synthesis in eukaryotic cells.

A Generalized Secondary Structure of tRNA Molecules

Figure 30.1 Generalized secondary structure of tRNA molecules. Circles represent nucleotides in the tRNA sequence. The numbers given indicate the standardized numbering for RNAs. Dots indicated places where the number of nucleotides may vary in different tRNA species.

30.1 The Genetic Code

• The genetic code is a triplet code, read from a fixed starting point in each mRNA.

• A group of 3 bases (a codon) codes for one amino acid.

• The code is not overlapping.• All the codons have meaning.• The genetic code is degenerate – in most cases,

each amino acid can be coded for by any of several triplet codons.

• The base sequence is read from a fixed starting point without punctuation.

• The genetic code is “universal”.

Features of the Code

Figure 30.2 (a) An overlapping versus a nonoverlapping code. (b) A continuous versus a punctuated code.

30.2 The Genetic Code

*Translation initiator as well as Met.

30.2 Matching an Amino Acid with Its Proper tRNA

• Codon recognition is achieved by aminoacyl-tRNAs.

• For accurate translation to occur, the appropriate aminoacyl-tRNA must “read” the codon through base pairing via its anticodon loop.

• The code by which each aminoacyl-tRNA synthetase matches up its amino acid with tRNAs constitutes a second genetic code.

• Aminoacyl-tRNA synthetases interpret the second genetic code.

• Aminoacyl-tRNA synthetases discriminate between the various tRNAs and amino acids.

Evolution Has Provided Two Distinct Classes of Aminoacyl-tRNA Synthetases

• Aminoacyl-tRNA synthetases fall into two fundamental classes on the basis of similar amino acid sequence motifs, oligomeric state, and acylation function.

• Class I aminoacyl-tRNA synthetases first add the amino acid to the 2'-OH of the terminal adenylate residue of tRNA before shifting it to the 3'-OH.

• Class II enzymes add it directly to the 3'-OH (see Figure 30.3).

Class I and Class II Synthetases

The Aminoacyl-tRNA Synthetase Reaction

Figure 30.3 (a) the overall reaction. (b) Aminoacyl-tRNA formation proceeds in two steps: (i) formation of the aminoacyl-adenylate and (ii) transfer of the activated amino acid to either the 2'-OH or 3'-OH.

The Aminoacyl-tRNA Synthetase Reaction

Figure 30.3

tRNA Molecules Have an Acceptor Stem at One End and an Anticodon at the Other

Figure 30.5 Ribbon diagram of the tRNA tertiary structure.

tRNA Recognition

For most tRNAs, a set of sequence elements is recognized by its specific aminoacyl-tRNA synthtase, rather than a single distinctive nucleotide or base pair.

Figure 30.6 Major identity elements in four tRNA species. Numbered filled circles indicate positions of identity elements within the tRNA that are recognized by its specific aminoacyl-tRNA synthetase.

Structure of an E. coli Glutaminyl-tRNA Synthetase Complexed with tRNA

Figure 30.7 Structure of E. coli glutaminyl-tRNAGln synthetase complexed with tRNAGln and ATP. The protein: tRNA contact region extends along one side of the entire length of this extended protein. The acceptor stem of the tRNA and the ATP (green) fit into a cleft at the top of the protein in this view. The enzyme also interacts extensively with the anticodon (lower tip of tRNAGln).

A Single G:U Base Pair Defines tRNAAlas

Figure 30.8 (a) a microhelix analog of tRNAAla is aminoacylated by alanyl-tRNAAla synthetase, provided it has the characteristic tRNAAla G3:U70 acceptor stem base pair.

30.3 Codon-Anticodon Pairing

Figure 30.9 Codon-anticodon pairing. Complementary trinucleotide sequence elements align in antiparallel fashion.

• Francis Crick proposed the “wobble” hypothesis for codon:anticodon pairing.

• He hypothesized that the first two bases of the codon and the last two bases of the anticodon form canonical Watson-Crick base pairs.

• But pairing between the third base of the codon and the first base of the anticodon follows less stringent rules.

• That is, there is play or wobble in base pairing at this position.



Figure 30.10 Pairing of anticodon inosine (I is on the left) with C, U, or A as the codon third base. Note that I is the keto tautomeric form.

Some Codons Are Used More Than Others

• Because more than one codon exists for most amino acids, variation in codon usage is possible.

• Thus the DNA of different organisms varies in relative A:T/G:C content.

• Even in organisms of average base composition, codon usage may be biased.

• Examples of nonrandom usage of codons in E. coli and in humans are given in Table 30.4.

• Preferred codons are represented by the most abundant isoacceptor tRNAs.

• And mRNAs for proteins that are synthesized in abundance tend to employ preferred codons.

Nonsense Suppression Occurs When Suppressor tRNAs Read Nonsense Codons

• Mutations that alter a sense codon to one of the three nonsense (stop) codons, UAA (ochre), UAG (amber), or UGA (opal), can result in premature termination of protein synthesis and the release of truncated (incomplete) proteins.

• It has been found that second mutations elsewhere in the genome can suppress the effects of nonsense mutations, so that the organism survives. This phenomenon is termed nonsense suppression.

• Suppressors are mutations in tRNA genes that change the anticodon so that the mutant tRNA is now able read a particular “stop” codon and insert an amino acid.

30.4 Structure and Assembly of Ribosomes

• Protein biosynthesis occurs by the process of translation on ribosomes.

• Ribosomes are compact ribonucleoprotein particles found in the cytosol of all cells.

• The E. coli ribosome is 25 nm diameter, 2520 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+.

• 30S subunit is 930 kD with 21 proteins and a 16S rRNA.

• 50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA.

• The 30S and 50S subunits combine to form the 70S ribosome.

30.4 Structure and Assembly of Ribosomes

* S proteins. † L proteins.Ribosomes are roughly 2/3 RNA. There are approximately 20,000 ribosomes in a cell representing 20% of cell's mass.

The rRNAs of E. coli Are Encoded by a Set of Seven Operons

Figure 30.11 E. coli has seven ribosomal RNA operons. Numerals to the right of the brackets indicate the number of species of tRNA encoded by each transcript.

Example: Pre-rRNAfrom one of theseven rRNA operons.

Prokaryotic Ribosomes Are Made from 50 Different Proteins and Three Different RNAs

• There is one copy of each ribosomal protein per 70S ribosome, except L7/L12 with 4. (L = large subunit)

• Proteins L7/L12 are identical except for extent of acetylation at N-terminus.

• Four L7/L12 plus L10 makes "L8".• Only one protein is common to large and small

subunits: S20 = L26. (S = small subunit)• The largest ribosomal protein is S1 (557 residues,

61.2 kD).• The smallest ribosomal protein is L34 (46 residues,

5.4 kD).

Ribosomes Self-Assemble Spontaneously in Vitro

• Ribosome subunit self-assembly is one of the paradigms for the spontaneous formation of supramolecular complexes.

• Structures of large and small subunits are known.• If individual proteins and rRNAs are mixed,

functional ribosomes will assemble.• rRNA acts as a scaffold on which the ribosomal

proteins convene in a specific order.• A tunnel runs through the large subunit .• Growing peptide chain is threaded through the

tunnel during protein synthesis.

The Overall Form of the 30S Structure is Essentially That of the rRNA

Structure of T. thermophilus ribosomal subunits and 70S ribosome. Features are labeled. (a) 30S; (b) 50S; (c) 70S; (d) side view of 70S.

Figure 30.13

Ribosomes Have a Characteristic Anatomy

• The details of ribosome structure have been revealed in recent years.

• The 30S subunit features a “head” and a “body”, from which a “platform” projects.

• The head, body, and platform create a cleft between them.

• The 50S subunit is a mitt-like globular structure with three distinct projections: a “central protuberance”, the “stalk”, and a ridge known as the “L7/L12 ridge”.

• The catalytic center on 50S, the peptidyl transferase, is located at the bottom of a deep cleft in 50S.

• Both subunits are involved in translocation, in which mRNA moves through the ribosome.

The Cytosolic Ribosomes of Eukaryotes Are Larger than Prokaryotic Ribosomes

• Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their prokaryotic origin.

• Eucaryotic cytoplasmic ribosomes are larger and more complex than procaryotic, but many of the structural and functional properties are similar.

• The structural organization of mammalian ribosomes is described in Table 30.6.

The Cytosolic Ribosomes of Eukaryotes Are Larger than Prokaryotic Ribosomes

30.5 Events in mRNA Translation

• All protein synthesis involves three phases: initiation, elongation, and termination.

• Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit.

• Elongation: Movement of ribosome along mRNA and synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. See Figure 30.14.

• Termination occurs when stop codon is reached. • Three tRNA molecules may be associated with the

ribosome:mRNA complex at any moment.

Figure 30.14 The basic steps in protein synthesis. The ribosome has three distinct binding sites for tRNA: the A, or acceptor, site; the P, or peptidyl, site; and the E, or exit, site.


Figure 30.15 The three tRNA binding sites on ribosomes. The view shows the ribosomal surfaces that form the interface between the (a) 30S and (b) 50S subunits in a 70S ribosome. The A (green), P (blue), and E (yellow) sites have bound tRNA.


Peptide Chain Initiation in Prokaryotes

• The components required for peptide chain initiation include (1) mRNA; (2) 30S and 50S ribosomal subunits; (3) a set of proteins known as initiation factors; (4) GTP; and f-Met-tRNAi

fMet.

• The initiator tRNA is f-Met-tRNAifMet, has formylMet.

• A formyl transferase adds the formyl group (see Figure 30.17).

• N-formyl-Met-tRNAifMet is only used for initiation,

and regular Met-tRNAmfMet is used for incorporation of

Met internally. • N-formyl methionine is first amino acid of all E.coli

proteins and the N-terminal Met is removed post-translationally in many proteins.

Initiator tRNA

Figure 30.16 The secondary structure of E. coli N-formyl-methionyl-tRNAi

fMet. The features distinguishing it from non-initiator tRNAs are highlighted.

The Transformylation of Methionyl-tRNAifMet

Figure 30.17 Methionyl-tRNAifMet formyl transferase catalyzes

the transformylation of methionyl-tRNAifMet using N10-formyl-

THF as formyl donor. The tRNA for reading Met codons within a protein (tRNAMet) is not a substrate for this transformylase.Eucaryotes do not use formyl-Met-tRNA.

mRNA Recognition and Alignment

• Correct positioning of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA.

• The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence, AGGA (see Figure 30.18). (No SD sequence in eucaryotes.)

• Initiation factor proteins, GTP, N-formyl-Met- tRNAifMet,

mRNA and 30S ribosome combine to form the 30S initiation complex.

• The 50S subunit then binds to form a 70S initiation complex.

• The initiation factors are soluble proteins required for assembly of these initiation complexes (Table 30.7).

Various Shine-Dalgarno Sequences Recognized by E. coli Ribosomes

Figure 30.18 These sequences lie about ten nucleotides upstream from their respective AUG initiation codon and are complementary to the UCCU core sequence element of E. coli 16S rRNA.

Properties of E. coli Initiation Factors

Table 30.8 Protein initiation factors.IF-2 ia a G-protein as are EF-Tu, EF-G and RF 3.

Events of Initiation

• 30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNAi

fMet (Figure 30.19).

• IF-2 delivers the initiator tRNA in a GTP-dependent process.

• Loss of the initiation factors leads to binding of 50S subunit.

• Note that the "acceptor site" is now poised to accept an incoming aminoacyl-tRNA.

The Sequence of Events in Peptide Chain Initiation

Figure 30.19 Initiation begins when a 30S subunit:(IF-3:IF-1) complex binds mRNA and a complex of IF-2, GTP, and f-Met-tRNAi

fMet.

Peptide Chain Elongation Requires Two G-Protein Family Members

The requirements for peptide chain elongation are:

(1) An mRNA:70S ribosome:peptidyl-tRNA complex (peptidyl-tRNA in the P site).

(2) Aminoacyl-tRNAs.

(3) A set of proteins known as elongation factors.

(4) GTP.

Peptide Chain Elongation Requires Two G-Protein Family MembersChain elongation can be divided into 3 steps:

(1) Codon-directed binding of the incoming aminoacyl-tRNA at the A site. Decoding center regions of 16S rRNA make sure the proper aminoacyl-tRNA is in the A site.

(2) Peptide bond formation: transfer of the peptidyl chain from the tRNA bearing it to the –NH2 group of the new amino acid.

(3) Translocation of the “one-residue-longer” peptidyl-tRNA to the P site to make room for the next aminoacyl-tRNA at the A site. These shifts are coupled with movement of the ribosome one codon further along the mRNA.


• Protein synthesis is vital to cell function, so elongation factors are present in significant quantities (EF-Tu is 5% of total protein in E. coli).

• EF-Tu binds aminoacyl-tRNA and GTP. • Aminoacyl-tRNA binds to A site of ribosome as a

complex with 2EF-Tu and 2GTP. • GTP is then hydrolyzed and the EF-Tu:GDP

complex dissociate. • EF-Ts is a guanine nucleotide exchange factor

(GEF) and it recycles EF-Tu by exchanging GTP for GDP.


Table 30.8 Protein elongation factors.

Peptidyl Transfer – the Central Reaction of Protein Synthesis• Peptidyl transfer, or transpeptidation, is the actual

peptide bond-forming step.• No energy input is needed here; the ester linking the

peptidyl moiety to tRNA is intrinsically reactive.• Figure 30.21 shows the location of the decoding

center and highlights the interface between a codon and anticodon.

• The peptidyl transferase, the activity catalyzing peptide bond formation, is associated with the 50S ribosomal subunit.

• The reaction is part of the 23S rRNA in the 50S.

Figure 30.20 The cycle of events in peptide chain elongation of E. coli ribosomes.

The structure at lower left is an EF-Tu:aminoacyl-tRNA complex.

The Decoding Center of the 30S Ribosomal Subunit is Composed Only of 16S rRNA

Figure 30.21 (a) The 30S subunit showing the region of the codon:anticodon association, as viewed from the 50S subunit.

The Decoding Center of the 30S Ribosomal Subunit is Composed Only of 16S rRNA

Figure 30.21 (b) 30S ribosomal subunit interactions with the codon-anticodon duplex during cognate tRNA recognition.

23S rRNA is the Peptidyl Transferase Enzyme

• This is the central reaction of protein synthesis. • 23S rRNA is the peptidyl transferase; this is a

ribozyme. It requires K+ but not ATP.• The peptidyl transferase center (PTC) of 23S

rRNA is shown in Figure 30.22 - these bases are among the most highly conserved in all RNA systems.

• No ribosomal proteins lie within 1.5 nm of the PTC, so none can participate in the reaction mechanism.

Catalytic Power of the Ribosome Depends on Tight Binding of Its Substrates

Figure 30.22 The peptidyl transferase active site.

Catalytic Power of the Ribosome Depends on Tight Binding of Its Substrates

• Bair-pairing interactions at the PTC lead to tight binding of the substrates (peptidyl-tRNA and aminoacyl-tRNA) such that the reactive groups are juxtaposed and properly oriented for reaction to occur.

• A network of H-bonds and electrostatic interactions stabilizes the highly polar transition state of the reaction (Figure 30.23).

• Translocation of peptidyl-tRNA from the A site to the P site follows the peptidyl transferase reaction (see Figure 30.20).

A Network of H bonds and Electrostatic Interactions Stabilizes the Transition State

Figure 30.23 The protein synthesis reaction proceeds via deprotonation of the α-amino group of the aminoacyl-tRNA, followed by nucleophilic attack of the α-amino on the peptidyl-tRNA carbonyl carbon to form the polar tetrahedral intermediate.

GTP Hydrolysis Fuels the Conformational Changes That Drive Ribosomal Functions

• Two GTPs are hydrolyzed for each amino acid incorporated into peptide.

• Hydrolysis drives essential conformation changes. • Total of four high-energy phosphate bonds are

expended per amino acid residue added - two GTP here and two in amino acid activation via aminoacyl-tRNA synthesis.

• Initiation is a one time expenditure of GTP.

Molecular Mimicry – The Structures of EF-Tu: Aminoacyl-tRNA, EF-G, and RF-3• EF-Tu and EF-G compete for binding to ribosomes.• EF-Tu can bind any aminoacyl-tRNA and deliver it to

the ribosome in a GTP-dependent reaction.• EF-G catalyzes GTP-dependent translocation.• The structure of the EF-Tu:tRNA complex is

remarkably similar to that of EF-G.• One view of early evolution suggests that RNA was

the primordial macromolecule, fulfilling all biological functions.

• The mimicry of EF-Tu:tRNA by EF-G may represent a fossil of early macromolecular evolution, when proteins took over the roles of RNA.

Molecular Mimicry – The Structures of EF-Tu: Aminoacyl-tRNA, EF-G, and RF-3

EF-Tu:tRNA EF-G RF-3

Peptide Chain Termination Requires a G-Protein Family Member

• Elongation continues until the 70S ribosome encounters a “stop” codon which signals chain termination.

• Proteins known as release factors recognize the stop codon at the A site and promote polypeptide release from the ribosome.

• Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier.

Termination: Release Factors “Read” the Stop Codon

Figure 30. The events in peptide chain termination.A stop codon (UAA, UAG or UGA) appears in the A site.

The 70S:RF-1:RF-3-GTP Termination Complex

Figure 30.24 The events in peptide chain termination.RF-1, RF-3 and GTP bind to the A site.

The Peptidyl Transferase Hydrolyzes the Ester Bond Linking Polypeptide to tRNA

Figure 30.24 The events in peptide chain termination. Peptidyltransferase acts as a hydrolase and the polypeptide is released from the ribosome.

Ribosome Recycling Factor Triggers Release of the Now-Uncharged tRNA

Figure 30.24 The events in peptide chain termination.70S ribosome dissociates from mRNA and separates into 30S and 50S subunits.

The Ribosome Subunits Cycle Between 70S Complexes and a Pool of Free Subunits

• Ribosome subunits cycle rapidly through protein synthesis.

• 80% of ribosomes are engaged in protein synthesis at any instant.

• When the 70S ribosome dissociates from mRNA, it separates into free 30S and 50S subunits.

• Intact 70S ribosomes are inactive in protein synthesis, because only 30S subunits can interact with initiation factors.

Figure 30.25 The ribosome life cycle. Note that IF-3 is released prior to 50S addition.

Ribosome Life Cycle

Multiple Ribosomes Can Translate the Same mRNA at a Given Time

Figure 30.26 Electron micrograph of polysomes – multiple ribosomes translating the same mRNA.

30.6 How Are Proteins Synthesized in Eukaryotic Cells

• Eukaryotic mRNAs are characterized by two post-transcriptional modifications:• The 5'-terminal 7methyl-GTP cap.• The 3'-terminal poly(A) tail.

• See Figure 30.27 for details.• The 5'-methyl-GTP cap is essential for mRNA

binding by eukaryotic ribosomes and also enhances stability of these mRNAs by preventing their degradation by 5'-exonucleases.

• The poly(A) tail enhances stability and translational efficiency of eukaryotic mRNAs.

The Characteristic Structure of Eukaryotic mRNAs

Figure 30.27 Untranslated regions ranging between 40 and 150 bases in length occur at both the 5'- and 3'-ends of the mature mRNA. An initiation codon at the 5'-end, invariably AUG, signals the translation start site.

Peptide Chain Initiation in Eukaryotes

• The events of eukaryotic peptide chain initiation are summarized in Figure 30.28.

• The properties of eukaryotic initiation factors (EIFs) are presented in Table 30.9.

• Initiation of protein synthesis in eukaryotes involves more than a dozen eukaryotic initiation factors.

• The initiator tRNA that carries only Met and functions only in initiation - it is called tRNAi

Met but it is not formylated.

Figure 30.28 The three stages in the initiation of translation in eukaryotic cells. See Table 30.9 for a description of the functions of the eukaryotic factors (eIFs).

Properties of Eukaryotic Translation Initiation Factors

Peptide Chain Initiation in Eukaryotes

• Eukaryotic protein synthesis is more complex than prokaryotic protein synthesis.

• It begins with formation of ternary complex of eIF-2, GTP and Met-tRNAi

Met.• This binds to 40S ribosomal subunit:eIF-1A & elF-

3: complex to form the 40S preinitiation complex. • Note no mRNA yet, so no codon association with

Met-tRNAiMet.

• mRNA then adds with several other factors, forming the initiation complex (Fig. 30.28).

• Note that GTP is required.• Proteins of the initiation complex scan to find the

first AUG (start) codon.

Figure 30.29 The 48S initiation complex. mRNA is shown as a black line, and the 5'-cap is a black circle. The coding region is shown as a yellow bar.

Control of Eukaryotic Peptide Chain Initiation & Regulation of Gene Expression

• Phosphorylation/dephosphorylation of translational components is a dominant mechanism for control of protein synthesis.

• Thus far, 7 proteins involved in initiation (4 initiation factors, two elongation factors, and ribosomal protein S6) are activated by phosphorylation.

• Phosphorylation of eIF-2a causes it to bind eIF-2B and ,rendering eIF-2 unavailable for protein synthesis (Figure 30.29).

α-Subunit Phosphorylation Controls the Function of eIF2

Figure 30.30 Control of eIF2 functions through reversible phosphorylation of a Ser residue on its α-subunit.

Inhibitors of Protein Synthesis

Two important purposes to biochemists

• These inhibitors (Figure 30.31) have helped unravel the mechanism of protein synthesis.

• Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics.

• Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth.

• Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis.

Some Protein Synthesis Inhibitors

End Chapter 30Protein Synthesis

reginald h. garrett charles m. grisham chapter 30 protein synthesis

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