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) 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 ( tRNAs vs. 20 AA)

Figure Genomes 3 (© Garland Science 2007) tRNA & protein synthesis. tRNAs 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) 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) 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 Genomes 3 (© Garland Science 2007) 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 Genomes 3 (© Garland Science 2007) Sedimentation coefficient by ultracentrifugation

Figure Genomes 3 (© Garland Science 2007) 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 Genomes 3 (© Garland Science 2007) (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 tRNA Met w/unmodified Met.

Figure 13.16a Genomes 3 (© Garland Science 2007) (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 tRNA Met ; 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) (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) (Cont.) Regulation of translation initiation. Global regulation transcript specific regulation Global regulation (e.g. under stressful conditions) by eIF-2 phosphorylation prevents GTP binding, therefore represses translation; transcript specific regulation by feedback inhibition or feedback activation mechanisms (left)

Figure Genomes 3 (© Garland Science 2007) (Cont.) Elongation P site (peptidyl site) A site (aminoacyl site) Large subunit has 2 sites P site (peptidyl site) w/tRNA Met ; A site (aminoacyl 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) (Cont.) Frame-shifting during elongation programmed frame-shifting Ribosome pauses spontaneously & moves back for 1 nt & continues translation: changes the reading frame; 3 types of frame-shifting: programmed frame-shifting enables translation of multiple proteins from the same mRNA

Figure 13.21b-c Genomes 3 (© Garland Science 2007) (Cont.) Frame-shifting during elongation Translation slippage translational bypass Translation slippage: enables a single ribosome to translate an mRNA that contains copies of 2 or more genes. Similarly, translational bypass.

Figure Genomes 3 (© Garland Science 2007) (Cont.) Termination a protein release factor ribosome release factor At the termination codon, A site is occupied by a protein release factor; ribosome disassociates by ribosome release factor (RRF).

Figure Genomes 3 (© Garland Science 2007) Post-translational processing Four major types of processing:

Figure Genomes 3 (© Garland Science 2007) (Cont.) Protein folding a dynamic process Four levels of protein structure; need correct tertiary structure to be activated; a dynamic process; for large proteins, renaturation is not always spontaneous due to (1) tendency to form insoluable aggregates; (2) more stable alternative folding pathways.

Figure Genomes 3 (© Garland Science 2007) (Cont.) Protein folding molecular chaperons & chaperonins Protein folding is assisted by molecular chaperons (to hold proteins in an open conformation for folding) & chaperonins (a protein complex to promote folding through a cavity & proof-read incorrectly folded proteins into correct folding).

Figure Genomes 3 (© Garland Science 2007) (Cont.) Proteolytic cleavage end-processing or poly-protein processing Protein cutting is either end-processing (to cut off N or C terminals to make functional proteins) or poly-protein processing (to cut into small pieces of functional proteins).

Figure Genomes 3 (© Garland Science 2007) (Cont.) Proteolytic cleavage end- processing An example of end- 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 Genomes 3 (© Garland Science 2007) (Cont.) Proteolytic cleavage poly- protein processing An example of poly- protein 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) (Cont.) Chemical modification

Figure 13.34a Genomes 3 (© Garland Science 2007) (Cont.) Chemical modification glycosylation More complex modification is glycosylation used to add large carbohydrate side chains to Serine (O- linked) or Asparagine (N- linked).

Figure Genomes 3 (© Garland Science 2007) (Cont.) Intein splicing intein homing 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 homing (convert a intein- gene into a intein+ gene; used as a mechanism to propagate).

Figure Genomes 3 (© Garland Science 2007) Protein degradation proteasome Proteolysis by proteases is dependant on degradation- susceptibility signals; in eukaryotes, proteasome unfolds proteins & cuts into 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.