8 Protein Synthesis, Processing, and Regulation. Chapt 8 Student learning outcomes Because proteins are the active players in most cell processes Explain general process of Translation of mRNA: indicate similarities, differences prokaryotes, eukaryotes
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Translation: synthesis of polypeptide
directed by mRNA template on ribosome
mRNAs read 5′ to 3′ direction;
polypeptide synthesized from NH2 to COOH terminus.
Amino acid specified by 3 bases (codon) in mRNA
rRNA, tRNA, mRNA roles
Translation is first step to form functional protein:
polypeptide chain must fold into appropriate conformation
often undergoes processing steps.
Gene expression is regulated at level of translation
Many controls on amounts and activities of proteins: ultimately regulates all aspects of cell behavior.
tRNAs (70 - 80 nt) align amino acids with codons on mRNA
Cloverleaf Structure; CCA at 3′ terminus,
anticodon loop binds to codon (complementary bp)
Aminoacyl tRNA synthetases
Attach amino acids to specific tRNAs
Each enzyme recognizes one amino acid,
as well as correct tRNA(s)
Costs ATP to attach
Ribosomes named according to sedimentation rates in ultracentrifugation
Ribosomes are abundant in cells
lot of protein synthesis;
E. coli ~ 20,000; mammalian cells ~ 10 x 106
Evidence rRNA does catalysis:
Noller et al. (1992): large ribosomal subunit could catalyze formation of peptide bonds even after 90% of ribosomal proteins were removed.
High-resolution structure of 50S
Ribosomal proteins absent from site
of peptidyl transferase reaction.
Ribosomal proteins mostly structural
Nobel Prize 2009
Translation initiates with Met, usually 5’-AUG.
Bacterial mRNA AUGs preceded by Shine-Dalgarno sequence - aligns mRNA on ribosome
Eukaryotic mRNAs recognized by 7-MeG cap at 5′ terminus.
Ribosomes scan downstream until initiation codon.
Translation: initiation, elongation, and termination.
Initiator met-tRNA and mRNA bind small ribosomal subunit.
Large ribosomal unit joins, forming functional ribosome.
Fig. 8.10 Bacterial initiation
Termination: elongation continues until stop codon (UAA, UAG, or UGA) translocated into A site.
Release factors recognize codons, terminate
Polysomes (polyribosome) :
mRNAs translated simultaneously by several ribosomes
Once ribosome moved from initiation site, another can bind.
Regulation of translation modulates gene expression:
translational repressor proteins
noncoding miRNAs, siRNA, RNAi
localization of mRNAs
Ex. Cis-acting sequence in mRNA binds repressor
Translation of ferritin mRNA regulated by repressor proteins.
Iron absent, iron regulatory protein (IRP) binds iron response element (IRE) in 5′ UTR, blocks translation
Iron present, get translation
Fig. 8.16 eukaryote
Fig. 8.18 mRNA
RNA interference (RNAi) short ds RNAs block translation
Small interfering RNAs (siRNAs)— ds RNAs, nuclease Dicer.
MicroRNAs (miRNAs)— transcribed by RNA pol II, cleaved by nucleases Drosha and Dicer.
RNA-induced silencing complex (RISC): siRNAs or miRNAs that pair perfectly induce cleavage of targeted mRNA; most miRNAs form mismatches, repress translation
Modification (phosphorylation) of initiation factors can regulate translation
global effects on overall translational activity
Ex. Phosphorylation of eIF2, eIF2B by protein kinases blocks exchange of bound GDP for GTP, inhibits initiation.
*2. Protein folding, processing is critical:
Polypeptide chains must undergo folding, other modifications, to become functional proteins
Information for conformation comes from amino acid sequence.
Folding and Processing includes:
S-S bonds between Cys residues
[Peptide bond isomerization (Pro residues)]
Proteolytic cleavage (removal of Met, pre-sequences)
Glycosylation (addition of sugars)
Addition of lipids
Chaperones: facilitate folding of other proteins.
Catalysts - facilitate assembly, are not part of complex.
Bind, stabilize unfolded or partially folded polypeptides
Protect chain from aberrant folding or aggregation until synthesis of an entire domain is complete
Stabilize unfolded polypeptide chains during transport into organelles; later assist refolding
Figs. 8.22, 23
Chaperones found as heat-shockproteins (Hsp)
Chaperonins -protein subunits in stacked rings
[Enzymes can be chaperones:
protein disulfide isomerase, peptidylprolylyisomerase]
Proteolytic processing - cleavage of polypeptide
1. Removes portions - initiator Met from NH2 terminus.
2. NH2-terminal signal sequencetargets protein for transport to specific destinations (details Chapt 10).
Signal sequence emerging from ribosome inserts into
membrane channel into ER (RER)
Signal sequence cleaved by protease (signal peptidase).
3. Proteolysis forms active enzymes or hormones by cleavage of precursors.
Glycosylationadds carbohydrate chains to proteins to form glycoproteins; occurs in ER and Golgi (Chapt. 10)
Carbohydrates: target proteins for transport to organelles, or secretion;
recognition sites in cell-cell interactions.
N-linked glycoproteins: carbohydrate attached to N atom in side chain of asparagine.
O-linked glycoproteins: carbohydrate attached to O atom in side chain of serine or threonine
Glycosylation starts in ER before complete translation
A 14-sugar oligosaccharide is transferred to an Asn residue of growing polypeptide chain.
Oligosaccharide assembled on lipid carrier (dolichol phosphate) on inner surface ER membrane.
Sugar chain in ER lumen
N-linked oligosaccharide modified by removal of three glucose residues, (further modifications in Golgi)
O-linked oligosaccharides added within Golgi, one at time
Sugar chain modifications
** Some eukaryotic proteins are modified with lipids, which often anchor them to plasma membrane.
N-myristoylationPalmitoylation * Prenylation * Glycolipids
N-myristoylation: myristic acid (14-carbon fatty acid) is attached to N-terminal glycine.
Proteins on inner face of plasma membrane
Fig. 8.33; 13.11
Src protein kinase
Prenylation: prenyl groups attached to S atoms in side chains of cysteine near C terminus.
Proteins involved in control of cell growth, differentiation,
ex Ras oncogene protein responsible for human cancers
Integral protein on inner surface plasma membrane
Fig. 8.34; 13.11
Ras G protein
Glycolipids (lipids linked to oligosaccharides)
added to C-terminal carboxyl groups
Anchor proteins to external face
of plasma membrane
Ex. Thy-1 on lymphocytes
GPI was added in lumen of ER
3* Regulation of protein function includes amounts and activities of proteins.
General mechanisms of control of proteins:
regulation by small molecules (allosteric)
Feedback inhibition is allosteric regulation:
regulatory molecule binds
enzyme site distinct from catalytic site
(allo= “other”; steric = “site”).
Cellular protein activities are regulated by GTP or GDP binding, including Ras oncogene proteins.
X-ray crystallography reveals
conformational differences Ras
of inactive GDP-bound (yellow, blue)
and active GTP-bound forms
Protein conformation determines
whether Ras binds target molecule,
signals cell to divide.
Mutations in RAS gene in ~20% of
human cancers: alter structure of Ras→
always active GTP-bound conformation,
continually signal cell division.
Fig. 8.38 (GTP is red)
Protein phosphorylation: reversible covalent modification activates or inhibits many proteins in response to environmental signals.
transfer phosphate groups
from ATP to OH groups of
side chains of ser, thr, or tyr.
Study by Ala substitutions:
Ala can’t get phosphorylated
Protein kinases in signal transduction pathways.
Sequential action: series of protein kinases transmits signal from cell surface to targets in cell;
Changes in cell behavior in response to environmental stimuli.
Signaling initiated by allosteric regulation – epinephrine (adrenaline) to cell surface, cAMP to kinase
Regulation by protein-protein interactions
Ex: inactive cAMP-dependent protein kinase
composed of 2 regulatory, 2 catalytic subunits
cAMP binds regulatory subunits:
conformational change dissociates complex.
Free catalytic subunits →
enzymatically active protein kinases.
cAMP is allosteric regulator →
alters protein-protein interactions.
4. Synthesis, degradation control protein levels:
Regulatory proteins short half lives: levels can change quickly
Faulty or damaged proteins recognized, rapidly degraded
major path eukaryotes
Ubiquitin conserved 76-aa peptide
Ubiquitin attaches to NH2-group
of Lys, then more to form chain
Specificity of enzymes controls
which proteins are degraded
Ex: Controlled degradation of cyclins,
Proteins regulate progression through cell cycle
Entry regulated by cyclin B
(regulatory subunit of
protein kinase Cdk1)
Cdk1 also activates
ubiquitin ligase that targets
cyclin B for degradation
at end of mitosis.
Inactivated Cdk1 →
cell enters interphase.
Protein degradation can also take place in lysosomes—membrane-enclosed organelles that contain digestive enzymes, including proteases.
Lysosomes digest extracellular proteins taken up by endocytosis; take part in turnover of organelles and proteins.
enclose small areas of
cytoplasm or organelles
You wish to express a cloned Eukaryotic DNA in bacteria. What type of sequence must you add for the mRNA to be translated on prokaryotic ribosomes?
You are interested in studying protein expressed on liver cells. How could treatment of these cells with a phospholipase (enzyme that cleaves phospholipids) enable you to determine whether protein is transmembrane or attached to cell surface by GPI anchor?
10. What is the function of 5’ and 3’ UTR of mRNAs?
12. Why is regulated proteolytic cleavage important for activity of certain proteins?