Cell Biology Chapter 8 wip

translation of mRNA

-read in 5’ → 3’ direction

-polypeptide chains synthesized from amino (N) to carboxy (C) terminus, occurs on ribosome

-codons in mRNA specify amino acids

transfer RNAs (tRNAs)

align amino acids with corresponding codons on mRNA template

-70-80 nucleotides long

aminoacyl tRNA synthetases

family of enzymes that attach amino acid to specific tRNA

-20 enzymes, each recognize single amino acid & correct tRNA

aminoacyl tRNA synthetases 2 step reaction

1) amino acid joined to AMP → aminoacyl AMP

2) amino acid transferred to 3’ CCA end of tRNA, AMP released

codon-anticodon base pairing

complementary base pairing aligns tRNA on the mRNA template

-40 different tRNAs, 20 different amino acids

-some tRNAs can recognize multiple mRNA codons (61 encode amino acids)

—wobble base

wobble base

-at third position in codon

-phenylalanyl tRNA base pairing (Guanosine-Uridine, GC)

-alanyl (Inosine-Uridine, IC, IA)

ribosomes

-designated by ultra-centrifugation sedimentation rates: 70S (bacterial) & 80S (eukaryotic)

-large & small subunits contain rRNA and proteins

-folding of rRNAs and proteins → distinct 3D structures

rRNAs use

complementary base pairing to form characteristic secondary structures

catalytic activity of rRNA

-large ribosomal subunit can catalyze formation of peptide bonds even after 90% of ribosomal proteins have been removed

-ribosomal proteins absent from site of peptidyl transferase rxn

-EPA (exit, peptidyl, aminoacyl

prokaryotic mRNA

polycistronic (multiple translation start sites)

eukaryotic mRNA

monocistronic (single translation start site)

translation of mRNA

-always starts with methionine (AUG) (N-formylmethionine in bacteria)

-translation initiation signals are different

mRNA translation in bacteria

-initiation codons are preceded by a Shine-Dalgarno sequence

-can initiate at the 5’ end of mRNA and at internal initiation sites of polycistronic mRNAs

Shine-Dalgarno sequence

aligns mRNA on ribosome

mRNA translation in eukaryotes

-eukaryotic mRNAs recognized by 7-methylguanosine cap at 5’ end

-40S subunit scans downstream of cap until it encounters the initiation codon

3 stages of mRNA translation

initiation, elongation, termination

initiation of mRNA translation in eukaryotes

methionyl tRNA (initiator) and mRNA bind to small subunit

-eIFs (eukaryotic initiation factors); 5’ cap and poly-A tail are recognized

-40S subunit scans for AUG → GTP is hydrolyzed & 60S subunit binds → forms 80S initiation complex

-internal ribosome entry site (IRE) recognized by eIF4G/eIF4A complex; recruit 40S ribosomal subunit w/ initiation tRNA/eIF2

elongation of mRNA in eukaryotes

large subunit joins → functional ribosome → elongation of polypeptide chain

-3 binding sites (EPA)

-initiator methionyl tRNA binds to P site

-elongation factor (eEF1α) brings next aminoacyl tRNa, binds to A site, pairs w/ second codon

-peptide bond formation catalyzed by rRNA in large subunit; methionine transferred to aminoacyl tRNA in A site (peptidyl tRNA in A, uncharged initiator in P)

-translocation requires eEF2 coupled to GTP hydrolysis

-ribosome moves 3 molecules (next codon in empty A site, peptidyl tRNA in P, uncharged tRNA in E)

-new aminoacyl tRNA binds in A → releases uncharged from E

-eEF1α released from ribosome bound to GDP, must be reconverted to GTP form (requires eEF1ßγ)

accuracy of elongation

-decoding center in small r subunit recognizes correct codon-anticodon base pairing → triggers conformational change (GTP hydrolyzed, EF-GDP released)

termination of mRNA translation in eukaryotes

elongation continues until a stop codon (UAA, UAG, UGA) reaches A site

-release factors (RF) recognize stop codons → terminate protein synthesis

simultaneous translation

one ribosome moves away from initiation site, then another can bind → polysome

polysome

group of ribosomes bound to an mRNA molecule

regulation of translation general mechanisms

translational repressor proteins, noncoding mRNAs, overall state of cell

regulation of translation by repressor protein

-ferratin (protein that stores iron)

-IRP (iron regulatory protein) binds to IRE (iron response element) if not enough protein

chaperones

proteins that facilitate folding of other proteins

-act as catalysts, bind to & stabilize unfolded/partially folded polypeptides

-stabilize unfolded polypeptide chains during transport into organelles → facilitate folding

-many initially identified as heat shock proteins

Heat shock proteins (Hsp)

-expressed in cells subjected to high temperatures

-stabilize and facilitate refolding of proteins that have been partially denatured

-Hsp 70 chaperones stabilize polypeptide chains during translation and transport (bind to short hydrophobic segments)

-polypeptide transferred to chaperonin for folding (subunits arranged in 2 stacked rings → form a double chambered structure)

protein misfolding diseases

caused by defects in protein folding

-misfolded proteins may form fibrous aggregates → amyloids

-ex. Alzheimer’s Disease (2 aggregates in brain: neurofibrillary tangles & amyloid plaques)

enzymes as chaperones

-protein disulfide isomerase (PDI)

-peptidyl prolyl isomerase

protein disulfide isomerase (PDI)

catalyzes disulfide bond formation

-abundant in ER (oxidizing environment allows S-S linkages common in secreted proteins)

peptidyl prolyl isomerase

catalyzes isomerization of peptide bonds that involve proline residues (also rate limiting step)

proteolysis

cleavage of a polypeptide chain removes portions from N-terminus

-initiator methionine

-may have N-terminal signal sequences (target the protein for transport to specific destination)

-signal sequence inserted into membrane channel as it emerges from ribosome → polypeptide chain passes through as translation proceeds → signal sequence cleaved by a membrane protease (signal peptidase)

proteolytic processing

formation of active enzymes or hormones by cleavage of larger precursors

glycosylation

adds carbohydrate chains to proteins → glycoproteins

-function: protein folding in ER, targeting proteins for transport, recognition sites in cell-cell interactions

N-linked glycoproteins

carbohydrate attached to N side chain of Asn

O-linked glycoproteins

carb attached to O side chain of Ser/Thr

N-linked glycosylation

starts in ER as translation occurs

-oligosaccharide is assembled on a lipid carrier (dolichol phosphate) in ER membrane → transferred to Asn

-sugar may be modified further

O-linked glycosylation

O-linked oligosaccharides added within the golgi apparatus

-formed by sequential addition of sugars

attachment of lipids to proteins

-N-myristoylation, prenylation, palmitoylation, glycolipids

N-myristoylation

myristic acid (14C FA) attached to an N-terminal Gly

-proteins associated w/ inner face of plasma membrane

prenylation

prenyl groups attached to S in Cys side chains near C terminus

-proteins involved in control of cell growth & differentiation

palmitoylation

palmitic acid (16C FA) added to S in internal Cys side chains

-important in association of some proteins w/ cytosolic face of PM

glycolipids

added to C-terminal carboxyl groups

-anchor proteins to external PM

-contain phosphatidylinositol → glycosylphosphatidylinositol (GPI) anchors

protein regulation by small molecules

change in enzyme conformation changes its activity (small molecules bind → conformational change)

-feedback inhibition

-some proteins regulated by GTP/GDP binding (ras oncogene proteins)

feedback inhibition

regulatory molecule binds to enzyme but not at catalytic site

-ex. of allosteric regulation

ras oncogene proteins (GTP vs GDP)

-ras-GTP can bind target molecule → signals cell division

-mutations can “lock” Ras in active conformation → continuous cell division

protein regulation by phosphorylation

-reversible modification; can activate or inhibit proteins in response to environmental signals

-catalyzed by protein kinases, reversed by protein phosphates

protein kinases

often components of signal transduction pathways

-ex. epinephrine signals breakdown of glycogen in muscle cells, catalyzed by glycogen phosphorylase, regulated by kinase)

protein regulation by other covalent modifications

-Acetylation of Lys

-Methylation of Lys and Arg

-Nitrosylation (addition of NO groups) to Cys

-Glycosylation of Ser and Thr

protein regulation by attachment of polypeptides

-addition of ubiquitin (or SUMO, ub-like) affects protein function

-ubiquitylation

ubiquitylation

Ub activated by E1 → transferred to ER, then complexes w/ E3 → E3 transfers Ub to protein

-can be reversed by deubiquitylation enzyme

regulation by protein-protein interactions

-interactions between protein subunits can regulate protein activity

-cAMP-dependent protein kinase

regulation of cAMP-dependent protein kinase

-inactive: 2 regulatory & 2 catalytic subunits

-cAMP binds regulatory subunits → conformational change → complex dissociated

-catalytic subunits are active protein kineases

protein degradation