protein synthesis 1 major topics covered: the genetic code trna: aminoacylation and base-pairing...

Protein Synthesis 1

Major topics covered:

•The genetic code•tRNA: aminoacylation and base-pairing•Ribosome structure/function: prokaryotic versus eukaryotic

related text:Biochemistry

Garret and Grisham, 4th ed.Chapter 30

contact info:David A. Schneider, Ph.D.

Department of Biochemistry and Molecular Geneticsdschneid@uab.eduoffice #: 934-4781

The Central Dogma of Biology:

DNA

RNA

protein

The Central Dogma of Biology:

DNA

RNA

protein

Othermacromolecules

The Central Dogma of Biology:

DNA

RNA

protein

A major molecular problem:How do you take a 4-base DNA/RNA

code and interpret the instructions to build proteins from a 20 amino acid

pool?

A major molecular problem:How do you take a 4-base DNA/RNA

code and interpret the instructions to build proteins from a 20 amino acid

pool?

rephrase:How do you translate the 4-base

DNA/RNA language into appropriate proteins?

Francis Crick proposed/predicted the Adaptor Hypothesis

– “…the RNA of the microsomal particles, regularly arranged, is the template”

– “…whatever went into the template in a specific way did so by forming hydrogen bonds”

– “…the amino acid is carried to the template by an adaptor...”

– “such adaptors…might contain nucleotides”

– “…a separate enzyme would be required to join each adaptor to its own amino acid…”

– “…the specificity required to distinguish between … isoleucine and valine would be provided by these enzymes”

Currently Known As:

mRNA

Codon-Anticodon Interactions

Aminoacyl-tRNA

tRNA

Aminoacyl-tRNA Synthetase

Editing by Aminoacyl-tRNA synthetases

Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.

Crick’s Predictions:

A visual model for the adapter hypothesis

Thus:•Genes are codes (recipes, in a way)•RNA polymerases copy the code into useful templates•Translation (a collaboration of tRNA and ribosomes) must crack the code correctly

The genetic code uses 3-base codons to generate 64 possible codon:anticodon interactions

(from the 4-base DNA/RNA sequence)

Different amino acids are encoded by one or more codons

tRNAs are the adapters that “crack” the triplet code and mediate the codon:anticodon pairing

Translation (on the surface) is very simple:•Charged tRNAs bind to the appropriate codons•Put a bunch in a row, according to the recipe in the mRNA•Bind all the amino acids together and, Wa-La!

Translation (on the surface) is very simple:•Charged tRNAs bind to the appropriate codons•Put a bunch in a row, according to the recipe in the mRNA•Bind all the amino acids together and, Wa-La!

There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame

Problem #1

Charging of the tRNA (ie. aminoacylation)

The amino acid is covalently attached to the 3’ “acceptor stem” of the tRNA by proteins called tRNA

synthetasestRNAGln bound to glutaminyl-tRNAGln synthetasetRNA cloverleaf diagram

Aminoacylation occurs by one of two pathways (class I or class II)

The interaction between the tRNA, the appropriate amino acid and the tRNA

synthetase is exceptionally important for translational fidelity

The structure of tRNAGln bound to its cognate tRNA synthetase demonstrates one mechanism for

specificity

Identity elements in tRNAs

Size of yellow ballis proportional to the fraction of 20 tRNA

acceptor types for whichthe nucleoside is an

observed determinant

Diagram of tRNA “identity elements”

tRNA synthetases can edit incorrect aminoacylation events as well

There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame

Problem #1 is solved!

How do the appropriate tRNAs bind to the correct triplet codon?

Codon : Anticodon binding specificity

Base pairing rules for the THIRD position of the codon

Illustration of non-specific interactions with inosine

Codon: 5’-CAC-3’Anticodon: 3’-GUG-5’

Codon: 5’-CAU-3’Anticodon: 3’-GUG-5’

A “wobble” example

There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame

Problem #1 is solved!and

Problem #2 is solved

How does translation choose the correct reading frame of the

triplet code?

The “reading frame” problem, illustrated:

In this case, the solution is easy:•Specific initiation of translation at a 5’ methionyl-

tRNA codon (AUG)•Strict, 3-nucleotide transitions during translation

elongation

There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame

All three problems are solved…

There are at least 3 major issues:1. Proper amino acid must be attached to every tRNA2. Proper binding of tRNA (anticodon) to mRNA (codon) must occur3. Triplet code must be interpreted in the proper frame

All three problems are solved…

Now:What molecular machine executes

the process of translation?

Topics covered in this portion of the lecture(the rest of today and Monday):

•Prokaryotic ribosome structure•Prokaryotic translation•Prokaryotic versus Eukaryotic:

Ribosome featuresTranslation mechanisms•Two examples of medical impact of translation

The ribosome and translation

The prokaryotic ribosome structure has been solved at atomic resolution

The bacterial ribosome is:•2 subunits (50S and 30S)•3 ribosomal RNAs (rRNAs)•52 proteins•Total Mass = ~2.5 million Daltons

Alberts 6-64d?

A low resolution “structure” to understand organization of sites in the ribosome

Alberts 6-64d?

A low resolution “structure” to understand organization of sites in the ribosome

How did the field progress from this cartoon to understanding molecular details of this massive cellular machine?

Early cryo-electron microscopy

experiments revealed the general shape of

the ribosome:led to initial

nomenclature

Better techniques led to better models:

Three dimensional model of the 70S ribosome

CP, central protuberenceSP, spur

Cate et al. (1999) Science 285:2097.

Better EM models permit visualization of the functional center of the 70S ribosome

Liljas (1999) Science 285:2077.

AminoacylPeptidyl

Exit

A PE

APE

Large (50S) Subunit

•Proteins-purple•23S rRNA-orange & white•5S rRNA (top)-burgundy & white•A site tRNA- green•P site tRNA- red

From Cech, Science 289: 878 (2000)

The crystal structure of the prokaryotic large subunit

Large (50S) Subunit

•Proteins-purple•23S rRNA-orange & white•5S rRNA (top)-burgundy & white•A site tRNA- green•P site tRNA- red

From Cech, Science 289: 878 (2000)

No protein sidechain atoms lies within 18 angstroms

of the peptidyl transferase site, so

ribosome is officially a ribozyme.

The crystal structure of the prokaryotic large subunit

Schluenzen et al., Cell 102: 615 (2000)

Head

Platform

Body

Foot

Shoulder

NoseThe small (30S) subunit:

•RNA = gold ribbon•Proteins = colored ribbons

The crystal structure of the prokaryotic small subunit

Functional sites mapped onto spacefill model of large and small subunits

Green = A siteBlue = P siteYellow = E site

2009 Nobel Prize in Chemistry was awarded for structural insights into ribosome function

-picture from New York Times

From left to right:Venkatraman RamakrishnanMRC Laboratory of Molecular Biology, Cambridge, United KingdomThomas A. SteitzYale University, New Haven, CT, USAAda E. YonathWeizmann Institute of Science, Rehovot, Israel

That is the ribosome.

Nest question:What is translation and how does

it work?

We will deal with that on Monday!

THE END

-any questions?