Because >99% of genes encode proteins, "gene expression" is nearly synonymous with protein synthesis

GENE EXPRESSION: 2 STEPS: 1. TRANSCRIPTION. 2. TRANSLATION

REQUIRES: mRNA, tRNAs, RIBOSOMES

GENETIC CODE: Information in mRNA read in units of 3 nucleotides = CODON

There are 20 amino acids, and hence the absolute minimum number of coding units would be 20.

Since there are 4 bases (A, G, C, T/U), 2 nucleotides would give 4 x 4 = 16 possibilities, obviously not sufficient for 20 amino acids. Hence, 3 nucleotides is the minimum number required: 4 x 4 x 4 = 64 possible codons. The codon is DEGENERATE, in that some amino acids are specified by multiple codons (up to 6 codons possible for a single amino acid).

SIMILAR TOPOGICAL PROBLEMS TO TRANSCRIPTION. START, STOP SIGNALS?

tRNAs are charged with appropriate AA using Aminoacyl tRNA Synthetases in 2-step reaction (bound intermediate)

1. ATP + AA --> aminoacyl-AMP + PPi

2. AA-AMP + tRNA --> AA-tRNA + AMP

Requires a composite signal: "Start Codon" and "Shine-Dalgarno, or Ribosome Binding Site"

Start Codons: AUG 90%

GUG 10%

UUG 1%

CUG/Others 0.1%

All START codons are read by a single mRNA in eubacteria: f-Met-tRNA = formyl-Methionine-tRNA.

Shine-Dalgarno//RBS sequence.

4-8 bp upstream from Start Codon is a purine-rich sequence (consensus = 5' AGGAGGU 3') that is complementary to the 3' end of the 16S rRNA (5' ACCUCCU 3') of the 30 S ribosome subunit. This sequence positions the ribosome to initiate translation (SEE p. 89 of textbook).

Steps in initiation:

1. 30S subunit binds IF3 (blocks binding of 50S subunit)

2. mRNA binds to 30S-IF3 complex by pairing of 16S 3' end with RBS sequence of mRNA

3. f-Met-tRNA binds to start codon with assistance of IF2-GTP complex

4. IF3 is released when f-Met-tRNA binds; replaced by IF1

5. 50S subunit binds; GTP--> GDP + Pi and IF1 and IF2 release. Initiator tRNA is positioned in the

"P SITE" = PEPTIDYL SITE

Initiation in eucaryotes requires 10 or more IFs!

More complex process, slower; no consensus RBS sequence

ARCHAEA: more similar to eucaryotes;

11 IF proteins identified in M. jannaschii

3-part RXN: 1. AA-tRNA binding

2. Peptide bond formation by peptidyl transferase

3. Translocation

2 GTPs hydrolyzed per peptide bond during elongation cycle

Steps in Elongation Cycle:

1A. AA-tRNA binding requires two Elongation Factors (EF-Tu and EF-Ts = Temperature-Stable and Temperature-Unstable), which form a complex.

1B. Binding of GTP to EF-Tu releases EF-Ts, which binds AA-tRNA

1C. AA-tRNA binds to "A-site"= "Acceptor Site" of ribosome; GTP is hydrolyzed to GDP and EF-Tu is released

2. Peptide bond formation is catalyzed by 23S rRNA component of the 50S subunit (PEPTIDYL TRANSFERASE)

3. Translocation is facilitated by GTP hydrolysis and EF-G (Elongation Factor G). tRNA originally in P-site moves to E (EXIT) site; tRNA originally in A-Site moves to P-site; A-site now empty for incoming AA-tRNA

3 termination codons = STOP TRANSLATION = UAA, UAG, UGA

No tRNAs for these codons; instead RF (Release Factor) protein binds.

RF1: recognizes UAA and UAG

RF2: recognizes UGA and UAA

RF3: enhances activity of RF1 & RF2

GTP hydrolysis occurs, and peptidyl transferase activity is stimulated causing release of protein from tRNA

Several E. coli proteins (e.g., formate dehydrognase) contain the unusual AA selenocysteine = Cysteine with Sulfur replaced by Selenium; synthesized from serine after attachment to tRNASec

How is this encoded in mRNA? The normal code allows only the basic 20 amino acids to be placed in proteins.

Context, as for "Start". Codon is UGA + an elaborate secondary signal--a 40-nt stem-loop recognized by a specific EF, SelB (homolog of EF-Tu). SelB binds stem-loop, GTP and and selenocysteinyl-tRNA.

Archaea: also have Selenocysteine in some proteins and appear to use UGA codon; mechanism probably similar.

Error rate in translation = approximately 1/5000

Could probably be improved, but would slow down rate of protein synthesis too much to allow rapid growth to occur

Most of the specificity arises from AA-tRNA synthetases. Reversible 1st step (formation of AA-AMP). Discrimination here can be 104

Methylation of tRNAs improves discrimination ability of the synthetases for the 2nd substrate

Some mistakes will occur during decoding, and misincorporation can lead to release of Protein-tRNA. Incomplete protein is degraded. Imagine that misincorporation were to lead to stalling of the ribosome and a halt in synthesis of that protein. Cure is now worse than disease, if cure were stalling and ribosome drop-off at each misincorporation. Genetic code is such that most missense changes will cause less than 50% loss of activity.