RU BL 424 - Protein synthesis, processing and regulation

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BL 424 Chapt 8: Protein synthesis, processing and regulation Student learning outcomes: Because proteins are the active players in most cell processes. 1. Explain the general process of translation (synthesis of polypeptide chain on ribosome), and indicate the similarities and differences between prokaryotes and eukaryotes. 2. State several mechanisms that regulate amount of translation: proteins binding the mRNA, subcellular localization of the mRNA, miRNAs, or polyA tail length. 3*. Explain that formation of a functional protein requires proper folding and processing of the polypeptide chain: involves sorting and transport; enzymes control folding and processing. 4*. Explain several mechanisms of regulating protein activity in response to external signals: reversible binding of small molecules, covalent phosphorylation, binding to other proteins; specific enzymes are involved (including kinases and phosphatases). 5*. Describe two mechanisms that regulate levels of proteins by degradation: the ubiquitin-proteosome pathway, and the lysosome. Important Figures: 1, 2*, 4*, 7*, 8, 9*, 12, 14, 15, 16, 17, 19, 20, 22*, 23*, 24, 25, 27*, 28, 29, 30, 31, 34*, 36, 39*, 40*, 42, 43*, 44, 45. Important Table: 1; All review questions at end of chapter Transcription and RNA processing are followed by translation, 1. Translation of mRNA is a highly conserved process Translation is the synthesis of a polypeptide from amino end to carboxy-terminal end: Figs. 8.1, 8.2 tRNAs carry (covalently) specific amino acids: bind with their anticodons to codons on the mRNA. Aminoacyl tRNA synthetases put correct aa on tRNA mRNA is translated from 5’ to 3’ on ribosomes in cytoplasm (rRNA plus proteins: 70S prokaryote 50S + 30S (23S, 5S rRNA; 16S rRNA) 80S eukaryote: 60S + 40S (28S, 5.8S, 5S; 18S rRNA) the rRNA is catalytic, not merely structural (Fig. 8.4) detailed crystal structure of ribosome The structure of mRNA includes AUG start codon (Met) untranslated regions (UTRs) and coding sequence(s) (Fig. 8.7*, 8.8): Prokaryotes have free 5’ end (5’-PO4), ribosome binding sequence (Shine-Delgarno) and are often polycistronic Eukaryotes have 5’ CAP structure, Ribosome scans to find 1st AUG sequence 3’ end has polyA tail Usually monocistronic Translation involves initiation, elongation and termination (Fig. 8.9*). Many different non-ribosomal protein factors (translational factors) are involved in the stages (Fig. 8.10, 8.11, Table 1). Initiation in eukaryotes is more complex (Fig. 8.11)Elongation requires GTP hydrolysis Special release factors recognize the stop codons (Fig. 8.14) release peptide chain, dissociate tRNA and mRNA from ribosome. Polysomes: (Fig. 8.15) a group of ribosomes bound to one mRNA molecule: Many polypeptides can be synthesized from one mRNA [Some antibiotics inhibit growth of cells by targeting different steps in translation] Translation of specific mRNAs can be regulated: Iron responsive element in ferritin (Fig. 8.16) Repressor proteins binding the mRNA (Fig. 8.17) Subcellular localization of mRNA (Fig. 8.18) Xenopus oocyte Non-coding microRNAs binding RISC complex target destruction, inhibition of mRNAs (Fig. 8.19) PolyA chain length can be regulated. *Phosphorylation of initiation factors (Figs. 8.20, 8.21) Active form binds GTP; inactive has GDP (eIF2) 2* Protein folding and processing are critical to proper function Linear polypeptide sequence has the information necessary for folding, but nascent polypeptide chain is subject to degradation, aggregation. Molecular chaperone proteins facilitate (catalyze): intracellular folding of polypeptide into proper 3-D structure. Chaperones stabilize partly folded structures on ribosomes to prevent degradation (Fig. 8.22). use ATP to bind, protect hydrophobic region Chaperones help with protein transport (Fig. 8.23*) Chaperones often increase after stress to help refold proteins (Fig. 8.24) (ex. heat-shock proteins Hsp and chaperonins Big hollow structure helps refold) Other enzymes assist with protein folding: Protein disulfide isomerase (PDI) assists with correct S-S bonds between Cysteine residues (Fig. 8.25). Peptidyl prolyl isomerase alters Proline conformation In the peptide bond (cis -> trans configuration; Fig. 8.26). [most peptide bonds use trans configuration of the aa]Protein cleavage. Some polypeptides get selective proteolysis: Cleavage of peptide bond Most proteins get 1st aa (Met) cleaved off; Ex. cleavage to activate pre-hormone. (Fig. 8.28 on insulin). ** Signal sequences target proteins to cellular compartments. Proteins destined for secretion enter the ER lumen because of an amino-terminal signal sequence; Signal sequences hydrophobic at N-terminus, translocate protein across membrane. (Fig. 8.27*) Signal sequence is later cleaved by a signal peptidase. Glycosylation. Some proteins are modified by addition of carbohydrates in the ER and Golgi: (Fig. 8.29). (more in Chapter 10). Especially secreted and plasma membrane proteins Sugars aid in sorting and transport of proteins N-linked carbohydrates are attached to Asparagine residues: A 14-sugar residue is attached to NH2 of Asn on the growing polypeptide chain in the ER. (Fig. 8.30) (this chain was assembled on a lipid carrier (dolichol phosphate) in the ER membrane. Further modifications of sugar chains occur in the Golgi (Fig. 8.31) including addition, removal of sugars. O-linked carbohydrates are attached to Serine or Threonine: Usually only a few sugars are added to the –OH group, sequentially, in the Golgi. (Fig. 32) N-acetyl galactosamine Lipids. Some proteins are modified by addition of lipids, especially proteins targeted to anchor in plasma membrane. N-myristolyation is addition of one 14-C myristic acid (fatty acid) to an N-terminal Glycine residue (Fig. 8.33) (the N-terminal Methionine was already removed). Typically seen on inner face of plasma membrane Prenylation is addition of prenyl group to S atom on Cysteine near the COOH end of polypeptide; (3 other aa are then removed: AAX). and methyl group added to COOH end. (Fig. 34*) Prenyl groups include 15-C farnesyl, 20-C geranyl. This modification occurs on the Ras


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