Lecture 4. Polypeptide Synthesis Overview

(1)

Lecture 4

Polypeptide Synthesis Overview

Polypeptide synthesis proceeds sequentially from N Terminus to C terminus.

Amino acids are not pre-positioned on a template. Proved by the classic experiment of Dintzis & coworkers in 1961. They synthesized hemoglobin in a test tube using a

reticulocyte (red blood cell) system. They initiated protein synthesis and then added H³- labeled leucine. They isolated hemoglobin, partially labeled with incorporated H³-leucine, and created peptide fragments (labeled a, b, c, d, e, f, g below) by digestion of

hemoglobin with trypsin. Then they analyzed peptide fragments to determine the relative amount of radioactivity. The following results were obtained:

http://bass.bio.uci.edu/~hudel/bs99a/lecture23/index.html (1 of 5)5/25/2007 9:36:32 AM

(2)

(3)

Figure 4.1.1: Incorporation of radioactive leucine into hemoglobin after different periods of incubation.

There is a gradient from the C-terminus to the N-terminus of the protein, which implies that synthesis takes place sequentially from N-terminus to C-terminus. As the time of incubation is increased, the total amount of radioactivity is increased but the gradient remains. Results are expressed as the ratio of an H³ label (tritium) in leucine to an internal control of C¹⁴. Data of Dintzis (1961).

(4)

Ribosomes read mRNA in the 5'->3' direction, not the 3'->5' direction.

To prove, one must know the Genetic Code. For example,

AAA

codes for Lys,

AAC

codes for Asn and

CAA

codes for Gln. In a cell-free protein synthesizing system, initiate protein synthesis by adding artificial mRNA, consisting of poly

A

with one

C

(cytosine) at the 3' end. Analyze the N and C termini of the peptide fragments.

Nucleotide Sequence Protein Sequence Direction of Synthesis

5'-AAAAAA(AAA)nAAC-

3' Lys-Lys-(Lys)n-Asn 5' -> 3'

3'-CAAAAA(AAA)nAAA-

5' Gln-Lys-(Lys)n-Lys 3' -> 5'

Next: Polypeptide Synthesis Overview (continued)

(5)

Please report typos, errors etc. by EMAIL (mention the title of this page).

(6)

Polypeptide Synthesis Overview (cont.)

Active translation occurs on polyribosomes

Electron micrographs of ribosomes actively engaged in protein synthesis revealed by

"beads on a string" appearance. This implied that each mRNA transcript is read

simultaneously by more than one ribosome. In fact, a second, third, fourth, etc. ribosome starts to read the mRNA transcript before the first ribosome has completed the synthesis of one polypeptide chain. Multiple ribosomes on a single mRNA transcript are called

polyribosomes or polysomes. The individual ribosomes are separated by gaps of 50 Å to 150 Å. Thus, there is approximately one ribosome per 80 nucleotides.

(7)

Chain elongation links the growing polypeptide to

incoming aminoacyl-tRNA

The newly formed polypeptide chain is attached to a tRNA molecule, not to a protein or mRNA molecule. To prove, use a cell-free protein synthesizing system with artificial mRNA, such as polyA. Add H³-Lys. Wash active ribosomes in high salt to dissociate and stop protein synthesis. Analyze results and find highest concentration of H³-Lys -- always with tRNA. From many such experiments, there are now known to be three tRNA binding sites on the ribosomes:

A Site The ribosomal site which binds the incoming aminoacyl-tRNA (Acceptor site).

P Site

The site which holds the peptidyl-tRNA, that is the tRNA which is covalently linked to the growing polypeptide chain (Peptidyl site).

E Site A site which transiently binds to the outgoing, deacylated tRNA (Exit site).

http://bass.bio.uci.edu/~hudel/bs99a/lecture23/lecture4_2.html (2 of 4)5/25/2007 9:36:34 AM

(8)

Three steps are involved in the synthesis of

one polypeptide chain

1. INITIATION

❍ Assembly of active ribosome and the reading of the first mRNA codon

(START codon).

❍ Occurs once per polypeptide chain.

2. ELONGATION

❍ Involves three distinct steps for each mRNA codon or amino acid in the new

(9)

polypeptide chain:

1. Transfer of aminoacyl-tRNA from cytoplasm to A-site of ribosome.

2. Covalent linkage of new amino acid to growing polypeptide chain - peptidyl transfer.

3. Movement of tRNA from A-site to P-site and simultaneous movement of mRNA by 3 nucleotides - translocation.

❍ Occurs multiple times per polypeptide.

3. TERMINATION

❍ Reading of final mRNA codon (STOP codon) and dissociation of polypeptide

from ribosome.

❍ Occurs once per polypeptide chain.

Next: Initiation of Protein Synthesis

(10)

Initiation of Protein Synthesis

INITIATION - Assembly of active ribosome and the reading of the first mRNA codon.

Occurs once per polypeptide chain.

The signals for initiation:

mRNA Start Codon

Prokaryotes:

AUG

(very rarely also

GUG

&

UUG

) Eukaryotes:

AUG

After protein is made, the N-terminal methionine is frequently cleaved off.

Shine-Dalgarno Sequence

To distinguish the start

AUG

codon from

AUG

codons which code for internal methionines or from a fortuitous

AUG

combination in another reading frame, the start

AUG

codon in prokaryotes is preceded by a highly conserved sequence at the 5´ end of the mRNA transcript which serves as a ribosome-binding site. The Shine-Dalgarno sequence is a purine-rich tract of 3-10 nucleotides and precedes the start

AUG

codon by approximately 10 nucleotides upstream on the 5´ side. The Shine-Dalgarno sequence forms base pairing with a highly conserved, pyrimidine-rich region of the 16S rRNA

(11)

which is part of the 30S ribosomal subunit. This base-pairing properly aligns the start

AUG

codon in the P site of the 30S ribosomal subunit. In

eukaryotes, protein synthesis usually begins with the first

AUG

codon from the 5´ end. Eukaryotic mRNA has a special cap at the 5´ end of the mRNA which is recognized by a cap-binding protein.

Special Initiator tRNA

Only one special tRNA is used to place the first amino acid, which is always a formyl-methionine (fMet):

(12)

The initiator tRNA is called fMet-tRNA. It differs from all other tRNAs by one less base pair in the acceptor stem region and by three consecutive G:C base pairs in the anticodon region. These changes alter the 3-dimensional structure of the fMet-tRNA in subtle ways.

fMet-tRNA^fMet and Met-tRNA^Met both recognize the

AUG

codon. A methionine is attached to both tRNAs by the same methionyl tRNA synthetase. However, once the methionine is attached to either tRNA, a special enzyme called formyl transferase, which recognizes only the initiator tRNA^fMet adds a formyl group to the amide nitrogen. Because the amide N is now blocked from further reaction, this amino acid can only be positioned at the start of a polypeptide chain.

Soluble Protein Factors

In prokaryotes, there are three initiation factors: IF1, IF2, IF3, named in order of discovery, not in order of their function on the ribosome. The initiation factors are proteins that facilitate the assembly of an active

(13)

ribosomal particle and the placement of the first fMet-tRNA^fMet. In eukaryotes, there are 10 initiation factors, called eIF1, eIF2, etc.

IF3 binds to 30S subunit and promotes the dissociation of the 30S subunit from the 50S subunit and the subsequent binding of the mRNA. IF1 helps IF3 by increasing the dissociation rate between the 30S and 50S subunit.

IF2, in complex with GTP, binds to fMet-tRNA^fMet and places it in the P-site of the 30S subunit. Then IF3 is released, causing GTP to hydrolyze to GDP, which in turn releases IF2-GDP. GTP hydrolysis promotes the release of IF1 and the subsequent association of the 30S subunit with the 50S subunit.

Next: Schematic of Initiation

(14)

Formation of the 70S initiation complex

Schematic of the initiation of protein synthesis in prokaryotes

(15)

1. Binding on IF1 & IF3 to an empty 70S ribosome dissociates it into 50S and 30S subunits.

2. a. 5' region of mRNA binds to free 30S ribsomal subunit, IF3 is released.

b. Initiator fMet-tRNA

^fMet

carried by IF2-GTP binds to P site of 30S ribsomal subunit.

3. Free 50S ribosomal subunit binds, IF1 & IF2-GDP + P

_i

are released.

Certain details of the process remain uncertain. For example, the exact order of binding of IF1, IF2, IF3, tRNA, and mRNA is unclear. The above is one current model (from the text book).

(16)

(17)

Schematic of the initiation of protein synthesis in eukaryotes

(18)

Note that the process is in principle very similar to prokaryotic initiation. Major differences from prokaryotic initiation are associated with the m⁷G cap of the mRNA and its interaction with the cap-binding protein (CBPI) and eIF4F (CBPII) instead of Shine-Dalgarno sequence- mediated alignment to properly position the first AUG codon in the P site of the ribosome.

Lecture 4. Polypeptide Synthesis Overview

Lecture 4

Polypeptide Synthesis Overview

Polypeptide synthesis proceeds sequentially from N Terminus to C terminus.

Ribosomes read mRNA in the 5'->3' direction, not the 3'->5' direction.

AAA

AAC

CAA

A

C

Next: Polypeptide Synthesis Overview (continued)

Polypeptide Synthesis Overview (cont.)

Active translation occurs on polyribosomes

Chain elongation links the growing polypeptide to

incoming aminoacyl-tRNA

A Site The ribosomal site which binds the incoming aminoacyl-tRNA (Acceptor site).

P Site

The site which holds the peptidyl-tRNA, that is the tRNA which is covalently linked to the growing polypeptide chain (Peptidyl site).

E Site A site which transiently binds to the outgoing, deacylated tRNA (Exit site).

Three steps are involved in the synthesis of

one polypeptide chain

Initiation of Protein Synthesis

INITIATION - Assembly of active ribosome and the reading of the first mRNA codon.

Occurs once per polypeptide chain.

The signals for initiation:

AUG

GUG

UUG

AUG

AUG

AUG

AUG

AUG

AUG

AUG

AUG

AUG

Next: Schematic of Initiation

Formation of the 70S initiation complex

Schematic of the initiation of protein synthesis in prokaryotes

1. Binding on IF1 & IF3 to an empty 70S ribosome dissociates it into 50S and 30S subunits.

2. a. 5' region of mRNA binds to free 30S ribsomal subunit, IF3 is released.

b. Initiator fMet-tRNA

carried by IF2-GTP binds to P site of 30S ribsomal subunit.

3. Free 50S ribosomal subunit binds, IF1 & IF2-GDP + P

are released.

Certain details of the process remain uncertain. For example, the exact order of binding of IF1, IF2, IF3, tRNA, and mRNA is unclear. The above is one current model (from the text book).

Summary

Polypeptide synthesis proceeds sequentially from N terminus to C

terminus

Ribosomes read mRNA in the 5'->3' direction, not the 3'->5' direction

Active translation typically occurs on polyribosomes

Chain elongation links the growing polypeptide to incoming

aminoacyl-tRNA

Three steps are involved in the synthesis of one polypeptide chain of

N residues:

1. INITIATION (1 x)

2. ELONGATION (N-1 x) 3. TERMINATION (1 x)

Initiation in prokaryotes requires:

1. One empty 70S ribosome

2. Three initiation factors: IF1, IF2, IF3

3. mRNA with Shine-Dalgarno sequence and START codon (AUG)

4. Initiator fMet-tRNA

5. One GTP

Initiation in eukaryotes is similar, but more complicated

Next lecture: Elongation & Termination of Protein Synthesis