Transcript ppt

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
• Explain that protein function requires proper folding
and processing, including sorting and transport
• Describe several mechanisms of regulation of protein
activity
• Describe 2 ways for protein degradation:
• Ubituitin-proteasome; lysosome
Introduction
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.
Translation of mRNA
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
.
Figs. 8.1,2
Translation of mRNA
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
Fig. 8.4
B, 30S,
C, 50S
Translation of mRNA
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
(Steitz, 2000):
Ribosomal proteins absent from site
of peptidyl transferase reaction.
Ribosomal proteins mostly structural
Nobel Prize 2009
ribosome structure
Fig. 8.6
Fig 8.7 Prokaryotic and eukaryotic mRNAs
mRNAs contain untranslated regions (UTRs 5’, 3’)
Eukaryotic mRNAs usually one polypeptide chain
• Monocistronic
Prokaryotic mRNAs often encode many polypeptides
• Polycistronic (e.g., lac operon).
Fig. 8.7*
Translation of mRNA
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.
Fig. 8.8
Translation of mRNA
Translation: initiation, elongation, and termination.
Initiator met-tRNA and mRNA bind small ribosomal subunit.
Large ribosomal unit joins, forming functional ribosome.
Fig. 8.9*
Many non-ribosomal proteins for initation.
Initiation starts with 30S ribosomal
subunit bound to IF1 and IF3.
mRNA, initiator N-formylmethionyl (fMet)
tRNA and IF2 (bound to GTP) join
IF1 and IF3 release, 50S subunit binds,
IF2-GDP is released
• 5’-CAU of tRNA binding 5’-AUG of mRNA
Fig. 8.10 Bacterial initiation
Fig 8.12 Elongation stage of translation
Elongation similar in prokaryotes and eukaryotes
• 3 binding sites: P (peptidyl), A (aminoacyl), and E (exit).
• Initiator met-tRNA bound at P site.
•
elongation factor (EF-Tu prokaryotes, eEF1a in eukaryotes)
bound to GTP brings aminoacyl tRNA to complex
Translocation –
• Ribosome moves 3 nt
• Next codon in empty A site.
• Peptidyl tRNA from A to P,
• Uncharged tRNA from P to E
• New aa-tRNA binds A site
• Uncharged tRNA leaves
Fig. 8.12
Fig 8.14 Termination of translation
Termination: elongation continues until stop codon
(UAA, UAG, or UGA) translocated into A site.
Release factors recognize codons, terminate
Fig. 8.14
termination
Translation of mRNA
Polysomes (polyribosome) :
mRNAs translated simultaneously by several ribosomes
Once ribosome moved from initiation site, another can bind.
begin synthesis
Fig. 8.15
polysomes
Translation of mRNA
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
Ferritin regulation
Fig 8.18 Localization of mRNA in Xenopus oocytes
Ex. Localization of mRNAs to specific regions of eggs
or embryos important in development:
permits proteins synthesized at appropriate sites.
•
•
•
Proteins binding 3′ UTRs can localize mRNAs to specific
regions of cells.
mRNAs with short poly-A tails are stored in oocytes;
translation activated at fertilization or later
Lengthening poly-A tails allows binding of poly-A binding
protein (PABP), stimulates translation
Fig. 8.18 mRNA
localization
Xenopus oocyte
Translation of 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
Fig. 8.19
RNAi
Translation of mRNA
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.
Fig. 8.20
Protein Folding and Processing
*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:
•
•
•
•
Chaperone proteins
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
Protein Folding and Processing
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
Fig 8.24 Sequential actions of chaperones
Chaperones found as heat-shock proteins (Hsp)
• Expressed in cells subjected to high temperatures.
• Stabilize, facilitate refolding of partially denatured proteins
Chaperonins - protein subunits in stacked rings
(double-chambered structure)
• isolates protein from cytosol, other unfolded proteins
Figs. 8.24
[Enzymes can be chaperones:
protein disulfide isomerase, peptidyl prolyly isomerase]
Protein Folding and Processing
Proteolytic processing - cleavage of polypeptide
1. Removes portions - initiator Met from NH2 terminus.
2. NH2-terminal signal sequence targets 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).
Figs. 8.27*
Fig 8.28 Proteolytic processing of insulin
3. Proteolysis forms active enzymes or hormones by
cleavage of precursors.
• Ex. Insulin synthesized as precursor polypeptide
• 2 cleavages (S-S bonds) produce mature insulin.
Fig. 8.28
Protein Folding and Processing
Glycosylation adds 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.
Fig. 8.29
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
Protein Folding and Processing
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.
Fig. 8.30
Sugar chain in ER lumen
Protein Folding and Processing
N-linked oligosaccharide modified by removal of three
glucose residues, (further modifications in Golgi)
O-linked oligosaccharides added within Golgi, one at time
Figs. 8.31,2
Sugar chain modifications
Protein Folding and Processing
** Some eukaryotic proteins are modified with lipids,
which often anchor them to plasma membrane.
• N-myristoylation
Palmitoylation
* 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
Lipid modifications;
Src protein kinase
Protein Folding and Processing
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
Lipid modifications;
Ras G protein
Protein Folding and Processing
Glycolipids (lipids linked to oligosaccharides)
added to C-terminal carboxyl groups
•
Anchor proteins to external face
of plasma membrane
• Contain phosphatidylinositol,
glycosylphosphatidylinositol
(GPI) anchors
Ex. Thy-1 on lymphocytes
GPI was added in lumen of ER
Fig. 8.36
glycolipid modifications
Regulation of Protein Function
3* Regulation of protein function includes
amounts and activities of proteins.
General mechanisms of control of proteins:
• regulation by small molecules (allosteric)
• phosphorylation
• protein-protein interactions
Feedback inhibition is allosteric regulation:
• regulatory molecule binds
enzyme site distinct from catalytic site
(allo = “other”; steric = “site”).
Fig. 8.37
Regulation of Protein Function
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)
Regulation of Protein Function
Protein phosphorylation: reversible covalent
modification activates or inhibits many proteins in
response to environmental signals.
• protein kinases:
transfer phosphate groups
from ATP to OH groups of
side chains of ser, thr, or tyr.
• protein phosphatases:
hydrolyze phosphorylated
amino acids
• Study by Ala substitutions:
Ala can’t get phosphorylated
Fig. 8.39
Regulation of Protein Function
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
Fig. 8.40
Regulation of Protein Function
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.
Fig. 8.42
Protein Degradation
4. Synthesis, degradation control protein levels:
• Regulatory proteins short half lives: levels can change quickly
• Faulty or damaged proteins recognized, rapidly degraded
Ubiquitin-proteasome pathway:
major path eukaryotes
• Proteasome degrades
polyubiquitinated proteins
• 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
Fig. 8.43
Protein Degradation
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.
Fig. 8.44
Protein Degradation
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.
Autophagy:
vesicles (autophagosomes)
enclose small areas of
cytoplasm or organelles
Recycle components.
Fig. 8.45
Review questions:
1. 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?
6. 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?