Protein Synthesis

 

I. Introduction

Translation from the four-letter nucleotide code of genetics converts the genetic message into the twenty-letter code of proteins.

This process involves the controlled action of some 150 macromolecules and requires one half of the bacterial cell's mass and 80% of its energy.

The unraveling of the mechanism of protein synthesis has been a major concern of biochemistry during my 50+ year involvement after receiving an undergraduate degree (1953).

During this time I have both created and collected schemes that summarize the process.

These are the skeleton of the intellectual framework on which I organize information concerning the process.

 

The cycle of an amino acid through protein and back to the amino acid can be divided into the following steps:

 

1. Activation of the amino acid and charging to tRNA.

2. Initiation of protein synthesis; formation of the initiation complex.

3. Elongation and the G cycle.

4. Termination of protein synthesis and release of the completed protein.

5. Posttranslational modification of the protein if required.

6. Degradation of the protein to the amino acids.

 

 

 

II. The Code

 

(see Stryer 5.5)

 

A. Introduction

1. Messenger RNA (mRNA), the blueprint for protein synthesis, is written in the language of ribonucleotides (A, G, C, U) and is read in blocks of three nucleotides that mean a particular amino acid or termination.

2. The code is redundant, unambiguous, and universal except for specific organelles or parasites.It is read in one direction.

 

B. Crick's adaptor hypothesis. (see Stryer 5.4.5).

1. A protein probably does not recognize the sequence of bases in the code.

2. Need another nucleotide to recognize - an adaptor.

3. The amino acid is covalently attached at the 3'-end of a tRNA molecule. 

 

C. mRNA (see Stryer 5.5.2)

1. Jacob and Monod postulated the existence of mRNA in the 1961 (publication date). They postulated that the messenger should have the following properties.
a. Be complementary to one strand of the DNA since it is copied from the genetic message in DNA.

b. Have a short half time because the proteins that are synthesized must be changed over time.

c. Should be of various sizes because proteins are of different sizes.

d. Should be associated with the ribosomes, the site of protein synthesis.

e. The messenger should be a nucleic acid.

2. Brenner, Jacob, and Meselson during the summer of 1961 found a nucleic acid with the properties listed above. Brenner had just won the Nobel prize. Volkin and Astrachan at Oak Ridge National Laboratory also found a short-lived RNA labeled with 32P.

 

D. Artifical mRNA used to decipher the code

1. General nature of the code
a. A group of three bases codes for one amino acid.

b. The code is not overlapping.

c. The base sequence is read from a fixed starting point. The mRNA sequences contain no commas.

d. The code is degenerate meaning in most cases that each amino acid may be specified by more than one triplet.

2. Marshall Nirenberg and Heinrich Matthaei used a polyuridylic nucleic acid as the template for the incoporation of phenylalanine into a polymer with the properties of a protein - trichloroacetic acid insoluble. Mixed ribonucleotide polymers yielded information concerning the potential code.

3. Nirenberg and Leder used ribonucleotide triplets of know sequence to bind a radioactive amino acid to ribosomes. Your instructor (Proc. Nat. Acad Sci., 53, 646-652 (1965) proposed an alternative method of using ribonucleotide triplet bound to cellulose, but our work was overshaddowed by Nirenberg's.

4. The resulting code dictionary (see inside back cover of Stryer, & Table 5.4, p. 134).

 

5. There can be editing of the mRNA so that the final message is not a contiguous set of bases in the original DNA. (see Stryer 5.6).

6. Wobble allows some tRNAs to recognize more than one codon. (see Stryer section 29.3.9).

 

 a. In the wobble hypothesis, the first two bases of the triplet form strong Watson-Crick base pairs.

b. When the first base of the anticodon is a U or G, the binding is less specific - wobble can occur - allowing recognition of up to three different triplets.

c. When an amino acid is specified by several different codons, the codons that differ in either the first or second bases require different tRNAs.

d. A minimum of 32 tRNAs are required to recognize all 61 codons specifying amino acids.

7. Overlapping genes in different reading frames are found in some viral DNAs. [fX-174]

 

III. The Ribosome

 

A. Site of Protein Synthesis

The protein synthesizing factory is the ribosome. Work by Paul Zamecnik using radioactive amino acids identified a ribonuclear protein fraction as the site of the earliest labeling. This fraction contains the ribosomes. The ribosome is a complex supramolecular machine. Each E. coli cell contains 15,000 or moe ribosomes. The ribosomes contain 65 % of the cell's RNA and 35 % of its protein.

 

B. Composition.

1. RNA
In bacteria 23S, 16S, and 5S.

In eukaryotes 28S, 18S, 5.8S, and 5S.

2. Protein
Bacteria large subunit 36, small subunit 21.

In eukaryotes large subunit ~49 and small subunit ~33.

3. Structure and summary

Structure of ribosomes now known in detail by X-crystallography.

4. Masses of components
Bacterial

Ribosome
Small subunit
Large subunit

70S, 2520 kD

30S, 930 kD

50S, 1590kD

RNA 1664 kD

16S, 1542 nt, 560 kD

1104 kD

23S, 2904 nt

5S, 120 nt

Proteins 857 kD

370 kD, 21 kinds

487 kD, 31 kinds
nt = nucleotide number

Rat liver

Ribosome
Small subunit
Large subunit

80S 4220 kD

40S 1400 kD

60S 2820 kD

RNA 2520 kD

18S, 1874 nt 700 kD

1820 kD

28SW, 4718 nt

5.8S, 160 nt

5S, 120 nt

Protein 1700 kD

700 kD 33 kinds

1000 kD 49 kinds

5. Ribosomes are self-assemblying.
There is an order of assembly that has been determined by Normua.

 

C. Ribosome are reused

In protein synthesis there is a ribosome cycle that is can in turn associate with different mRNAs to form different proteins. The ribosomal subunits are reusable and not for the synthesis of a specific protein.

 

D. Protein synthesis facts

1. Howard Dintzis showed in 1961 that synthesis was from N-terminus to C-terminus.

2. mRNA is read from in 5' to 3' direction.

3. Charles Yanofsky (1964) showed that genes are colinear with their specified proteins. Changes in the base sequence produce changes in the amino acid sequence in the same order: A, B, C, D; a, b, c, d.

4. In bacteria protein synthesis occurs on polysomes, i.g., tandemly arranged multiple ribosomes attached to a single messenger RNA meaning that as many copies of a protein can be made as there are ribosomes.

 

 

IV. Overview of Protein Synthesis

 

A. Stages of Protein Synthesis

 

1. Stage 1: Activation of amino acids
a. Two fundamental chemical requirements
(1) the carboxylic group of each amino acid must be activated in an ATP-requiring reaction to facilitate the formation of the peptide bond.

(2) a link must be established between each new amino acid and the information that specifies that particular amino acid in the mRNA.

b. Each of the 20 amino acids is covalently attached to a specific tRNA (transfer RNA, formerly known as sRNA for soluble) at the expense of ATP energy, using a Mg2+-dependent activating enzyme called amino acyl tRNA synthetases. When an amino acid is attached to a tRNA, the tRNA is charged.

2. Stage 2: Initiation

a. The mRNA bearing the coded information for the sequence of amino acids is bound to the smaller of the two ribosomal subunits and to the initiating tRNA. The initiating tRNA is most often N-formyl-methionyl-tRNA coded for by AUG.

b. The larger ribosomal subunit then binds to form the initiation complex. The process requires GTP and three cytosolic protein factors called inititation factors (IF-1, IF-2, & IF-3).

3. Stage 3: Elongation

a. The nascent polypeptide chain is lengthened by adding one amino acid at a time with each amino acid carried by its tRNA.

b. Movement of the mRNA is required to shift from one code word to the next. The hydrolysis of GTP powers the reaction and movement.

c. Elongation protein factors called EFs (EF-Tu, EF-Ts, & EF-G) are required. There is cycyling of these factors and the hydrolysis of GTP.

4. Stage 4: Termination

a. The completion of a polypeptide is signalled when a terminating codon is reached.

b. The peptide is then released from the ribosoome using release protein factors called RFs (RF1, RF2, & RF3).

5. Stage 5: Folding and posttranslational processing

a.Enzymatic processing may occur which may include removal of some of the N-terminal amino acids and covalent modification such as phosphorylation or attachement of a cofactor.

b. The polypeptide fold to the correct structure often aided by protein factors (chaperones).

 

B. Components of Protein Synthesis

 

 

V. Amino Acid Activation and Charging of tRNA

 

A. tRNA
1. Transfer RNAs are relatively small (~70 nt; 73-93 nt) single stranded RNAs that fold into a precise 3-dimensional structure. MW is 24 - 31 k.

2. Mitochondria and chloroplasts contain distinctive somewhat smaller tRNAs.

3. The 2-D structure is that of a cloverleaf. (Stryer 29.1.2)

4. 3-D structure is a L shape of tRNA seen in crystals. (Stryer 29.1.3)

 

B. Activating Enzymes
1. Reaction - two steps Stryer 29.2.1)

amino acid + ATP <===> aminoacyl-tRNA + PPi

aminoacyl-tRNA + tRNA<===> aminoacyl-tRNA + AMP

2. 3-D structure of an aminoacyl-tRNA synthetase.

3.There are about 800 molecules of each enzyme per cell or 1 for each 20 ribosomes.

4. Two classes of aminoacyl-tRNA synthetases. (Stryer 29.2.5)

 

C. A Second Genetic Code

1. The adaptor hypothesis just moved the recognition of a nucleotide sequence by an enzyme from one arena to another.

2. The bases now known to be involved in the recognition.

Positions in blue are the same in all tRNAs. Those involved with one recognition are shown in orange and for more than one in green.

 

D. Proofreading (Stryer 29.2.2 & 29.2.3)
1. The aminoacyl-tRNA synthetase enzyme can achieve proof-reading accuracy by using two different binding sites for the amino acid prior to its attachment to the tRNA.

2. The aminoacyl-tRNA synthetases can hydrolyzed the charged tRNA and the reaction is more likely to occur if an incorrect amino acid is attached to the tRNA.

3. The mistake rate is 1 in 104. If only we could achieve that accuracy as students and teachers.

 

E. General Structure of Amionoacyl-tRNAs

 

 

 

 

 

VI. Initiation

 
A. Initiating Codon
1. Protein synthesis starts at the N-terminal amino acid and proceeds to the C-terminal residue (Dintzis, 1961).

2. It was a surprise when met was found to be the N-terminal amino acid for at least 85% of the newly synthesized proteins (Waller) .

3. N-Formylmethinoyl-tRNA is the initiating aminoacyl-tRNA. AUG is the code. (Stryer 29.4.1)

4. Reaction

methionine + tRNAfMet + ATP ---> Met-tRNAfMet + AMP + PPi

 

B. Three steps of initiation. (Stryer Fig. 29.27)

1. Step 1.
The 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. IF-3 prevents binding of the 50S subunit.

The initiating codon, AUG, is guided into its correct position by the Shine-Dalgarno sequence in the mRNA.

Bacterial ribosomes have three sites that bind aminoacyl-tRNAs.

a. Aminoacyl or A site.

b. Peptidyl or P site.

c. Exit or E site.

2. Step 2.

The complex that contains the 30S ribosomal subunit, IF-3, and mRNA is joined by GTP-bound to IF-2 and the initiating fMet-tRNAfMet and the codon and anticodon base pair.

3. Step 3.

The complex then combines with the 50S ribosomal subunit, simultaneously the GTP is hydrolyzed to GDP and Pi which are released from the complex.

All initiation factors depart.

The initiation complex is thus formed.

 

C. The Signals

D. Initiation in Eukaryotes

 

E. The Protein Factors Required

 

 

VII. The Elongation Cycle

 

A. Requirements
1. The initiation complex described before.

2. Aminoacyl-tRNAs.

3. Three protein elongation factors (EF-Tu, EF-Ts, and EF-G. (see Stryer Fig. 29.28)

4. GTP.

 

B. Elongation Steps (Stryer 29.4.2)

1. Step 1: Binding an incoming aminoacyl-tRNA.

2. Elongation Step 2: Peptide bond formation

The peptidyl transferase is a ribozyme in the 23S RNA.

 

In elongation amino acids are added one at a time as called for by the next codon on the mRNA.

3. Elongation Step 3: Translocation (Stryer 29.4.3)

 

 

 

4. Proofreading on the ribosome
The identity of the amino acid on the tRNA is not checked by the ribosome. In fact the amino acid attached to a tRNA can be chemically modified, but the modified amino acid is incorporated according to the anticodon. (Stryer 29.3.8)

The proofreading that is done by the ribosome is only for the codon-anticodon interaction. This occurs during the EF-Tu GTPase action.

 

 

 

VIII. Termination and Release (Stryer 29.4.4)

 

A. Terminating Codons

 

1. UAA

2. UAG

3. UGA

4. Often there are two stops signs in a row.

 

B. Termination or Release Factors (Fig. 29.31 & 29.32)

1. The terminating codon occupies the ribosomal A site.

2. The release factors, proteins RF1, RF2, & RF3 accomplish:

a. hydrolysis of the terminal peptidyl-tRNA bond.

b. release of the free polypeptide and the last tRNA, now uncharged, from the P site

c. dissociation of the 70S ribosome into the 50S and 30S subunits so that they may be used in a new cycle of polypeptide synthesis

3. The specific functions of each RF have not been established.

 

C. Energy Cost of Protein Synthesis

1. Forming the aminoacyl-tRNA requires two equivalents (ATP --> AMP + PPi).

2. GTP is cleaved during the first elongation step.

3. Another GTP is cleaved during the translocation step.

4. Sum thus more than 4 high energy phosphate bonds are cleaved during protein synthesis. The high energy cost is to have a particular amino acid next to another particular amino acid in an informed sequence.

 

D. Polysomes - 10 to 100 ribosomes on a single mRNA

 

E. Coupling of transcription and translation occurs in bacteria.

 

 

IX. Processing and Folding

The last step in protein synthesis involves posttranslational modification in which the protein achieves its final biologically active conformation and additional groups.

 

A. Amino-terminal and Carboxyl-terminal Modifications

1. Often the N-terminal formyl-N-methionyl residue and some number of following residues are removed. In 50 % of eukaryotic proteins the N-terminus is acetylated.

2. Sometimes the C-terminal amino acid is enzymatically removed.

 

B. Loss of Signal Sequences

1. The "zip codes" that direct proteins to certain cellular sites and contain 15-30 amino acid residues at the N-terminus.

2. Protein targeting will be discussed later.

 

C. Modification of Individual Amino Acids

1. The -OH groups of ser, thr, & tyr may be enzymatically phosphorylated using ATP.

2. An extra carboxyl group may be added to a glu, for example, the blood-clotting protein prothrombin.

3. Monomethyl and dimethyl lys residues may be formed.

4. Lysine and proline may be hydroxylated.

 

 

D. Attachment of Carbohydrate Side Chains
1. Glycoproteins are formed by the attachment of carbohydrate residues.

2. They can be N- or O-linked.

 

E. Addition of Isoprenyl Groups

1. Isoprene modification.

2. Ras protein and lamins.

 

 

F. Addition of Prosthetic Groups (covalently)

1. Biotin, heme

2. Lipoic acid

3. Flavin

 

G. Proteolytic Processing

1. Blood-clotting

2. Zymogen activation

3. Preproinsulin to insulin

 

H. Formation of Disulfide Cross-links

1. Cys + Cys --> cys-S-S-cys

 

I. Folding

1. Energy and other proteins may be involved. (see Stryer 3.6)

 

 

X. Inhibitors of Protein Synthesis

 

A. Puromycin Disrupts Formation of the Peptide Bond

 

B. Tetracyclins Inhibit Bacterial Protein Synthesis
1. Block the A site and block the binding of aminoacyl t-RNAs.

 

C. Chloramphenicol

1. Blocks peptidyl transferase

2. Bacteria, mitochondria, and chloroplasts.

 

D. Cycloheximide

1. Blocks peptidyltransferase in 80S ribosomes but not 70S: eukaryote but not bacterial.

 

E. Streptomycin (against bacteria)

1. Misreading of the genetic code at low concentrations.

2. Blocks initiation at high concentrations.

 

F. Diphtheria Toxin

1. Catalyzes the ADP-ribosylation of diphthamide (a modified histidine) in eukaryotic eEF2.

 

G. Ricin

1. Toxic protein from castor beans.

2. Inactivates the eukaryotic 60S ribosomal subunit bu depurinating a specific adenosine in 23S rRNA.

 

 

 

XI. Protein Targeting

 

A. Eukaryotic Cells Have Many Compartments and Structures
1. These have specific functions and thus require specific proteins.

2. There is a mechanism for sorting and directing particular proteins to particular sites. I like to think of it as zip codes.

3. There are short sequences of amino acids that serve as signal sequences - the zip code.

 

B. Posttranslational Modification of Many Eukaryotic Proteins Begins in the Endoplasmic Reticulum

1. Lysosomal, membrane, or secreted protein targeting begins in the ER.

2. Signals vary from 13 to 36 amino acid residues

a. About 10 to 15 hydrophobic amino acid residues.

b. One or more positively charged residues

c. A short cleavage sequence often including ala.

3. Signal reconition particle (SRP)
a. The signal sequence and the ribosome are bound by the particle.

b. GTP is bound and the elongation of the peptide halts at about 70 amino acids.

c. Goes to the cytosolic face.

d. The complex is delivered to a peptide translocation complex.

e. SRP then dissociates and hydrolysis of GTP occurs.

f. Elongation of the peptide now resumes.

g. The signal sequence is removed by a signal peptidase within the ER lumen.

 

 

C. Glycosylation Plays a Key Role in Protein Targeting

 

 

1. Lysosomal targeting

 

2. Phosphorylation of a mannose residue on lysosome-targeted enzymes.

 

D. Proteins are Targeted to Mitochondria and Chloroplasts by Similar Pathways.

 

 

E. Nuclear Targeting

 

 

F. Bacteria Also use Signal Sequences

 

Export signal

 

 

G. Cells Import Proteins by Receptor-mediated Endocytosis

 

 

XII. Protein Degradation

 

A. Ubiquitin Marks Proteins for Degradation

 

B. Degradation Occurs in the Proteasome

 

 

Powerpoint presentation

 

Protein synthesis movie

 

Web sites

 

Protein synthesis

THCME
http://www.indstate.edu/thcme/mwking/protein-synthesis.html

KUMC

http://www3.kumc.edu/jcalvet/bioc801h/

Animation

http://www.ncc.gmu.edu/dna/ANIMPROT.htm

http://www.biostudio.com/demo_freeman_protein_synthesis.htm

http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/protein_synthesis/protein_synthesis.html

rpi

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/translate.htm

 

 

02/28/05