Chapter 13:Synthesis and Processing

of Proteome

Copyright © Garland Science 2007

Transcriptome expression

• Synthesis of the proteome (tRNAs decode, polymerization in ribosome) • Processing of the proteome (folding, cutting, chemical modifications)

• Degradation of the proteome.

Figure 13.1 Genomes 3 (© Garland Science 2007)

13-1. tRNA & protein synthesis.

tRNAs are adaptor molecules between mRNA and polypeptide

Both physical (amino-acylation) & informational (codon-anticodon recognition)

Isoaccepting tRNAs specific for same AA (30-50 tRNAs vs. 20 AA)

Figure 13.2-3 Genomes 3 (© Garland Science 2007)

13-1. tRNA & protein synthesis.

tRNAs 74-90 nt in length; cloverleaf structure.

Acceptor arm attaches amino acid; anticodon arm attaches mRNA; 3 other arms are conserved.

Some positions are completely invariant; important for tertiary structure stability.

Figure 13.4 Genomes 3 (© Garland Science 2007)

13-1. tRNA & protein synthesis.

Aminoacyl-tRNA synthetase catalyzes transfer of amino acid to 2’ or 3’ –OH of tRNA

20 synthetases in Class I (2’-OH) & Class II (3’-OH) highly specific to amino acids.

Figure 13.6 Genomes 3 (© Garland Science 2007)

13-1. tRNA & protein synthesis.

Attachment of tRNA to mRNA is based on codon-anticodon interactions by base-pairing.

Wobble effect due to the curved shape of anti-codon may allow non-standard base pairing (e.g. G-U & 3’-UAI-5’ in bacteria; 16 of 48 human tRNAs read 2 codons).

Figure 13.12 Genomes 3 (© Garland Science 2007)

13-2. Ribosome in protein synthesis.

E. coli has 20,000 ribosomes in cytoplasm; human has even more; complex of rRNAs + proteins

Functions include to coordinate protein synthesis by placing mRNA, tRNA, proteins in correct positions; catalyze some translation reactions.

Figure 13.10 Genomes 3 (© Garland Science 2007)

Sedimentation coefficient by

ultracentrifugation

Figure 13.14 Genomes 3 (© Garland Science 2007)

13-2. Ribosome in protein synthesis.

In E. coli, ribosome is assembled on mRNA at initiation codon w/translation initiation factor IF-3 (prevents premature dissociation); 3’ of 16S rRNA attached to ribosome binding site (Shine-Dalgarno sequence).

Figure 13.15 Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Translation initiation in bacteria.

Initiation codon AUG (Methionine); initiator tRNA is modified by attaching –COH to Met N terminal (fM); IF-2 & GTP are used by large subunit to bind; internal AUG is recognized by a different tRNAMet w/unmodified Met.

Figure 13.16a Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Translation initiation in eukaryote.

Most mRNAs don’t contain ribosome binding sites (unlike bacteria); preinitiation complex (40S) is first assembled prior to binding; eIF-2 binds GTP & unmodified tRNAMet; cap binding complex acts as a bridge in between; binding also affected by poly(A) via PADP, a poly(A) binding protein.

Figure 13.16b Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Translation initiation in eukaryote.

Preinitiation complex scans along mRNA until it reaches the initiation codon (a few tens or hundreds nt downstream & located within Kozak consensus sequence); large subunits then attach.

Figure 13.17a Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Regulation of translation initiation.

Global regulationGlobal regulation (e.g. under stressful conditions) by eIF-2 phosphorylation prevents GTP binding, therefore represses translation; transcript transcript specific regulationspecific regulation by feedback inhibition or feedback activation mechanisms (left)

Figure 13.18 Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Elongation

Large subunit has 2 sites P P site (peptidyl site) site (peptidyl site) w/tRNAMet; A site (aminoacyl A site (aminoacyl site)site) w/tRNA for the next codon.

Elongation factor EF-1 ensures accuracy of new tRNAs; peptidyl transferase forms new peptide bond; EF-2 translocates the new tRNA & opens up A site.

Figure 13.21a Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Frame-shifting during elongation

Ribosome pauses spontaneously & moves back for 1 nt & continues translation: changes the reading frame; 3 types of frame-shifting: programmed programmed frame-shifting frame-shifting enables translation of multiple proteins from the same mRNA

Figure 13.21b-c Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Frame-shifting during elongation

Translation slippageTranslation slippage: enables a single ribosome to translate an mRNA that contains copies of 2 or more genes. Similarly, translational bypasstranslational bypass.

Figure 13.22 Genomes 3 (© Garland Science 2007)

13-2. (Cont.) Termination

At the termination codon, A site is occupied by a protein a protein release factorrelease factor; ribosome disassociates by ribosome release ribosome release factorfactor (RRF).

Figure 13.24 Genomes 3 (© Garland Science 2007)

13-3. Post-translational processing

Four major types of processing:Four major types of processing:

Figure 13.25-26 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Protein folding

Four levels of protein structure; need correct tertiary structure to be activated; a dynamic a dynamic processprocess; for large proteins, renaturation is not always spontaneous due to (1) tendency to form insoluable aggregates; (2) more stable alternative folding pathways.

Figure 13.27-28 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Protein folding

Protein folding is assisted by molecular molecular chaperons chaperons (to hold proteins in an open conformation for folding) & chaperonins& chaperonins (a protein complex to promote folding through a cavity & proof-read incorrectly folded proteins into correct folding).

Figure 13.29 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Proteolytic cleavage

Protein cutting is either end-processing end-processing (to cut off N or C terminals to make functional proteins) or or poly-protein poly-protein processingprocessing (to cut into small pieces of functional proteins).

Figure 13.31 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Proteolytic cleavage

An example of end- end-processing processing is pre-pro-insulin. Step 1. Cut off 24 amino acids from N terminal to give pro--insulin; step 2. Cut internal B chain to give insulin.

Figure 13.32 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Proteolytic cleavage

An example of poly- poly-protein processingprotein processing used as a way to reduce size of genomes w/ a single gene & 1 promoter & 1 terminator; can be spliced in various ways in different cells.

Table 13.6 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Chemical modification

Figure 13.34a Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Chemical modification

More complex modification is glycosylation glycosylation used to add large carbohydrate side chains to Serine (O-linked) or Asparagine (N-linked).

Figure 13.35 Genomes 3 (© Garland Science 2007)

13-3. (Cont.) Intein splicing

A protein version of RNA splicing (vs. extein); first discovered in yeast in 1990; also found in bacteria & archaea; most ~150 aa & self-catalyzed; intein homingintein homing (convert a intein- gene into a intein+ gene; used as a mechanism to propagate).

Figure 13.37 Genomes 3 (© Garland Science 2007)

13-4. Protein degradation

Proteolysis by proteases is dependant on degradation-susceptibility signals; in eukaryotes, proteasomeproteasome unfolds proteins & cuts into 4-10 aa; released to cytoplasm & further broken down to individual amino acids.

Chapter 13 Summary

End result of genome expression is proteome (a collection of proteins in a cell); tRNA 3’ end is attached to amino acid by aminoacylation; 5’ end is attached to mRNA by condon-anticodon interactions; wobble effect allows single tRNA read more than 1 codons.

Bacterial ribosome has internal binding site for mRNA; eukaryote doesn’t; initiation is controlled by global or transcript-specific mechanisms; unusual elongation includes programmed reading frame-shifting and translation bypassing; proteins are processed by proteolytic cleavage or chemical modifications & degraded by proteasome.