Lecture 25: Protein SynthesisKey learning goals:• Be able to explain the main stuctural features of

ribosomes, and know (roughly) how many DNA and protein subunits they contain.

• Understand the main functions of the big subunit.• Understand the main functions of the lile subunit.• Explain what a polysome is.• Understand how ribosomes place themselves on the start

codon. This is different in bacteria and in eukaryotes. Be able to compare and contrast these mechanisms.

• Understand what bacterial IF-1 and IF-3 do. • Understand what the eukaryotic eIF4 complex does.• Understand what a polycistronic mRNA is. Be able to

explain why polycistronic mRNA’s are very common in bacteria, and extremely rare in eukaryotes.

• Be able to explain why coupled transcription and translation occur in bacteria, but not in eukaryotes.

• Understand the special role of formylated methionine (fMet) in bacterial initiation.

Initiation. Locate the starting point on the template; assemble the polymerization machinery.

Elongation. Add a protomer to the growing polymer, as specified by the sequence on the template; repeat many times.

Termination. Cease elongation; disassemble the elongation hardware.

Template-mediated polymer synthesis: 3 stages

TRANSLATION PARTS LIST:

• mRNA — the template• amino acids (20)• tRNAs (approximately 40)• aminoacyl tRNA synthases (20)• ATP• GTP• ribosome -small subunit (decoding center) -large subunit (peptidyl transferase center)• initiation factors• elongation factors• termination factors

Ribosomes & Protein SynthesisThis diagram summarizes a lot of what you will need to know about bacterial protein synthesis.

Venki Ramakrishnan

erythrocyte 8,000 nm

bacterium 2,000 nm

300 bp DNA 2 nm wide 100 nm long

ribosome 25 nm

= 10 nm = 100 Å

David Goodsell

prokaryotes eukaryotesmakes the

polypeptide

decodes themRNA

makes thepolypeptide

decodes themRNA

Harry Noller

Noller Group, UCSC • http://rna.ucsc.edu/rnacenter/ribosome_rht.html

The 70 S bacterial ribosome

Ribosomal proteins lie mainly on the surface

Crystal Structure of the Eukaryotic 40S Ribosomal Subunit in Complex with Initiation Factor 1 Julius Rabl, et al. Science 331, 730 (2011)

The folding of ribosomal subunits is highly conserved

Evolutionary conservation & divergence of ribosomal proteins

Rabl et al., Science 331:730

Proteins of the small subunit. Cores found in all kingdoms are light blue. Archaeal cores are orange. Proteins or extensions uniquely found in eukaryotes are red.

Distinctive features of the eukaryotic ribosome map to the cytoplasmic surface

Ben-Shem et al., Science 334:1524

Eukaryote-specific RNA loops in red; eukaryote-specific proteins in yellow.

40 S 60 S

Conserved region surrounding polypeptide exit tunnel

100 nm

mRNA with multiple translating ribosomes: a polysome

100 nm

5´

3´

mRNA with multiple translating ribosomes: a polysome

Three Stages in Translation

Initiation: the ribosome is placed on the start codonElongation: mRNA-templated polypeptide polymerizationTermination: the polypeptide and mRNA are released

Note: this cartoon applies to bacteria. In eukaryotes, transcription occurs in the nucleus, translation in the cytoplasm. But the directions are the same in all cases.

AUG

In bacterial initiation, ribosome small subunit binds directlyto Shine-Delgarno initiation sites on the mRNA

In eukaryotic initiation, the small subunit binds the 7-methyl-G cap, then scans 5´ to 3´ to find a start codon

The ribosome has three tRNA binding sites:

1. Aminoacyl-tRNA2. Peptidyl-tRNA3. Exit

To understand initiation, you first must understand the basics of elongation!

APE

Direction oftRNA and mRNAmovement throughribosome

Three major steps in elongation:

A site:tRNA selection

P site:peptidyl transfer

translocation:uncharged tRNAexits from E site

Note that the growing polypeptide chain is transferred onto the incoming aa-tRNA!

The aa on the incoming aa-tRNA is not transferred onto the chain!

Initiation in bacteria

Ingredients:mRNAfMet-tRNA[fMet]initiation factors

IF-1IF-2IF-3

GTP and Mg2+

Small (30 S) subunit

AUG start codon aligned in the P site

Shine-Delgarno sequence bindssmall subunitRNA

IF-1 occupiesthe small subunit’sA site

Bacteria use a specialized initiator tRNA charged with a modified amino acid, N-formylmethionine (fMet).

Eukaryotes use plain old Met.

Initiation in bacteria

fMet

The presence of peptides containing N-terminal fMet is interpreted by animal immune systems as a sign that bacteria are present or that mitochondria have ruptured.

In other words, for us fMet is a danger signal.

Starting state for elongation:• Initiation factors have fallen off• Large (50S) subunit bound• fMet-tRNA and AUG codon are in P site• A and E sites empty

Initiation in bacteria

Eukaryotic initiation: the small subunit scans from 5´cap until it finds a start codon

As a consequence, eukaryotic mRNAs are almost always monocistronic: they contain only a single initiation site and encode only one polypeptide.

✁ ✁In some cases, a polyprotein can be cleaved by site-specific proteases to yield more than one polypeptide

monocistronicAUG

AUG AUG AUG

polycistronic

Initiation in eukaryotes

Ingredients:mRNAMet-tRNAinitiation factors:

eIF2 > know what this doeseIF4 > know what this doeseIF5eIF6

GTP, ATP, and Mg2+40 S subunit

eIF4 complex binds mRNA 5´ cap & poly-A tail

AP

Initiation in eukaryotes

Small subunit scans for AUG ATP consumption!

not fMet

Starting state for elongation• eIF4 complex stays bound to 5´ cap• Other initiation factors have fallen off• Large (60S) subunit bound• Met-tRNA and AUG codon are in P site• A and E sites empty

Initiation in eukaryotes