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3 PROTEIN
Adenine (A)
Thymine (T)
5-Methylcytosine (5mC)
Guanine (G)
Cytosine (C)
Nucleoside
[structure of deoxyadenosine]
Nucleotide
Nomenclature
Nucleoside +deoxyribose
Nucleotide +phosphate
adenosine guanosine
inosine
ii). Structure the Structure of theof DNA DNA doublechain helix polynucleotide
Double-stranded DNA
5 3
B DNA
3 5 3 5
Chemistry of DNA
Forces affecting the stability of the DNA double helix
hydrophobic interactions - stabilize - hydrophobic inside and hydrophilic outside stacking interactions - stabilize - relatively weak but additive van der Waals forces hydrogen bonding - stabilize - relatively weak but additive and facilitates stacking electrostatic interactions - destabilize - contributed primarily by the (negative) phosphates - affect intrastrand and interstrand interactions - repulsion can be neutralized with positive charges (e.g., positively charged Na+ ions or proteins)
Denaturation of DNA
Double-stranded DNA Strand separation and formation of single-stranded random coils
Hyperchromicity
Absorbance maximum for single-stranded DNA Absorbance Absorbance maximum for double-stranded DNA
220
260
300
The absorbance at 260 nm of a DNA solution increases when the double helix is melted into single strands.
50
0 50 70 Temperature oC 90
50
60
70 Temperature oC
80
Average base composition (G-C content) can be determined from the melting temperature of DNA
k2
Slower, rate-limiting, second-order process of finding complementary sequences to nucleate base-pairing
% DNA reassociated
Kinetic fractions:
fast intermediate slow
50
Cot1/2
slow (single-copy)
100
I I I log Cot
106 copies per genome of a low complexity sequence of e.g. 300 base pairs
1 copy per genome of a high complexity sequence of e.g. 300 x 106 base pairs
high k2
low k2
Features Includes most genes 1 Interspersed throughout genome between and within genes; includes Alu sequences 2 and VNTRs or mini (micro) satellites Highly repeated, low complexity sequences usually located in centromeres and telomeres
Satellite (tandem) 0
~10%
fast ~10% intermediate ~15% Alu sequences are about 300 bp in length and are repeated about 300,000 times in the genome. They can be found adjacent to or within genes in introns or nontranslated regions.
2
50
slow (single-copy) ~75% 100
1 Some
genes are repeated a few times to thousands-fold and thus would be in the repetitive DNA fraction
TTAGGGTTAGGGTTAGGGTTAGGG
plants algae
insects mollusks bony fish amphibians reptiles
The size of the human genome is ~ 3 X 109 bp; almost all of its complexity is in single-copy DNA.
birds
The human genome is thought to contain ~30,000 to 40,000 genes. 104 105 106 107 mammals 108 109 1010 1011
Gene structure
promoter region exons (filled and unfilled boxed regions)
5
translated region
The (exon-intron-exon)n structure of various genes histone total = 400 bp; exon = 400 bp
b-globin
total = 1,660 bp; exons = 990 bp HGPRT (HPRT) total = 42,830 bp; exons = 1263 bp
From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.
5 6
X
4 Alu 5 Alu 6
Chromatin structure
Nucleosome structure
Nucleosome core (left) 146 bp DNA; 1 3/4 turns of DNA DNA is negatively supercoiled two each: H2A, H2B, H3, H4 (histone octomer) Nucleosome (right) ~200 bp DNA; 2 turns of DNA plus spacer also includes H1 histone
Histones (H1, H2A, H2B, H3, H4) small proteins arginine or lysine rich: positively charged interact with negatively charged DNA can be extensively modified - modifications in general make them less positively charged
Phosphorylation Poly(ADP) ribosylation Methylation Acetylation Hypoacetylation by histone deacetylase (facilitated by Rb) tight nucleosomes assoc with transcriptional repression Hyperacetylation by histone acetylase (facilitated by TFs) loose nucleosomes assoc with transcriptional activation
Nucleofilament structure
Telomeres are protective caps on chromosome ends consisting of short 5-8 bp tandemly repeated GC-rich DNA sequences, that prevent chromosomes from fusing and causing karyotypic rearrangements.
telomere
centromere
telomere
telomere structure
<1 to >12 kb
(TTAGGG)many (TTAGGG)few senescent young
most differentiated somatic cells have decreased levels of telomerase and therefore their chromosomes shorten with each cell division
G0
Quiescent cells
G1
phase
phase
phase
G2
Mitosis
5 3 5 3
3 5 3 5
Initiation of DNA synthesis at the E. coli origin (ori) origin DNA sequence 5 3 A A A 3 5 binding of dnaA proteins
A A A
A
A
A
A A
A
A
B C
A A A
B C
A
A A A A A A
G
RNA primer
B C
B C
DNA polymerase
5 5 RNA primer 3
5 3
3 5
5 3
3 5
5 3
3 5
3
...has to be discontinuous. This is the lagging strand.
replication for
5 3
3 5
5 3
3 5
lagging strand (synthesized discontinuously) The leading and lagging strand arrows show the direction
of DNA chain elongation in a 5 to 3 direction The small DNA pieces on the lagging strand are called Okazaki fragments (100-1000 bases in length)
Strand separation at the replication fork causes positive supercoiling of the downstream double helix 3 5 5 3
breaks and reseals the DNA to introduce negative supercoils ahead of the fork Fluoroquinolone antibiotics target DNA gyrases in many gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)
3 5
5 3
5 3
RNA primer
RNA primer 5
pol III
DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer 3 5 5
pol I
DNA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously removing the RNA primer with its 5 to 3 exonuclease activity
DNA ligase seals the gap by catalyzing the formation of a 3, 5-phosphodiester bond in an ATP-dependent react 5
3 5
5 3
Single-strand binding protein (SSB)
C B
pol III
DNA gyrase - this is a topoisomerase II, which breaks and reseals double-stranded DNA to introduce negative supercoils ahead of the fork pol I
Rep protein SSB DNA pol III DNA pol I DNA ligase
DNA polymerase III is the main replicating enzyme DNA polymerase I has a role in replication to fill gaps and excise primers on the lagging strand, and it is also a repair enzyme and is used in making recombinant DNA molecules
Mutation
Mechanism Frequency____ chromosome 10-2 per cell division missegregation (e.g., aneuploidy)
base pair mutation 10-10 per base pair per (e.g., point mutation, cell division or or small deletion or 10-5 - 10-6 per loc insertion generation
Many polymorphisms exist in the genome the number of existing polymorphisms is ~1 per 500 bp there are ~5.8 million differences per haploid genome polymorphisms were caused by mutations over time polymorphisms called single nucleotide polymorphisms (or SNPs) are being catalogued by the Human Genome Project as an ongoing project
CATTCACCTGTACCA GTAAGTGGACATGGT
CATCCACCTGTACCA GTAGGTGGACATGGT
CATGCACCTGTACCA GTACGTGGACATGGT
CATCACCTGTACCA GTAGTGGACATGGT
base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine deletion insertion
CATGTCACCTGTACCA GTACAGTGGACATGGT
Spontaneous mutations can be caused by tautomers Tautomeric forms of the DNA bases
Adenine
Cytosine
AMINO
IMINO
Guanine
Thymine
KETO
ENOL
Cytosine
Guanine
Cytosine
Adenine
C G
C G C A
C G
C A
tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands
T A
which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired
Chemical mutagens
O
N NH NH N NH2
guanine
8-oxyguanine (8-oxyG)
the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation mutation of the MTH1 gene causes increased tumor formation in mice
Mechanisms of Repair
cytosine
uracil
5-methylcytosine
thymine
More than 30% of all single base changes that have been detected as a cause of genetic disease have occurred at 5-mCpG-3 sites
Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 100
Life span
10
RNA
Adenine Cytosine Guanine Uracil (U)
Secondary structure
Tertiary structure
translated region
3 AAU termination
The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis
Transcription
initiation elongation
termination
RNA product
mRNA
PuPuPuPuPuPuPuPu AUG
[
-30 region TTGACA AACTGT -36 -31
-30
Promoter
-10 +1
]
5 mRNA
+20
T T G AC A
82 84 79 64 53 45%
TATA AT
79 95 44 59 51 96%
consensus sequences
a b (Rifampicin target) b s
2 1 1 1
uncertain forms phosphodiester bonds binds DNA template recognizes promoter and facilitates initiation
a2bbs
holoenzyme
a2bb
core polymerase
sigma factor
closed promoter complex (moderately stable) the sigma subunit binds to the -10 region
once initiation takes place, RNA polymerase does not need very high affinity for the promoter sigma factor dissociates from the core polymerase after a few elongation reactions
elongation takes place with the core RNA polymerase The sigma cycle
RNA
A=T
RNA
A = T
U=A
U=A
RNA synthesis usually initiated with ATP or GTP (the first nucleotide) RNA chains are synthesized in a 5 to 3 direction
promoter binds CAP and RNA polymerase operator binds the lac repressor promoter - operator lac I P O lac Z lac Y lac A
lac repressor
LACTOSE
b-galactosidase
b-galactosidase
permease
acetylase
GLUCOSE + GALACTOSE
the function of the lactose (lac) operon is to produce the enzymes required to metabolize lactose for energy when it is required by the cell
Regulation of the lactose operon - negative control promoter - operator lac I P O lac Z lac repressor
RNA polymerase from binding to the promoter
lac Y
lac A
lac I
lac Z
lac Y
lac A
RNA pol
allolactose
lac I
lac Z
lac Y
lac A
lac I
lac Z
lac Y
lac A
NO TRANSCRIPTION
repressor (with bound allolactose) dissociates from the op negative control (repression) is alleviated, however...
lac I
lac Z
lac Y
lac A
NO TRANSCRIPTION
RNA pol
Regulation of the lactose operon - positive control in the presence of both lactose and glucose it is not necessary
for the cell to metabolize lactose for energy in the absence of glucose and in the presence of lactose it becomes advantageous to make use of the available lactose for energy in the absence of glucose cells synthesize cyclic AMP (cAMP) cAMP1 serves as a positive regulator of catabolite operons (lac operon) cAMP binds the dimeric cAMP binding protein (CAP)2 binding of cAMP increases the affinity of CAP for the promoter binding of CAP to the promoter facilitates the binding of RNA polymerase
1 cAMP = 3, 5 cyclic adenosine monophosphate
active CAP
+
lac I P O lac Z lac Y lac A
NO TRANSCRIPTION
lac I
RNA pol
lac Z
lac Y
lac A
lac repressor
b-galactosidase
permease
acetylase
inactive repressor the function of the lactose (lac) operon is to produce the enzymes
required to metabolize lactose for energy when it is required by the cell
6. RNA Processing
a). Steps in mRNA processing i). Capping ii). Cleavage and polyadenylation iii). Splicing b). Chemistry of mRNA splicing c). Spliceosome assembly and splice site recognition i). Donor and acceptor splice sites ii). Small nuclear RNAs d). Mutations that disrupt splicing e). Alternative splicing
Steps in mRNA processing (hnRNA is the precursor of mRNA) capping (occurs co-transcriptionally) cleavage and polyadenylation (forms the 3 end) splicing (occurs in the nucleus prior to transport)
exon 1 intron 1 exon 2
Capping occurs co-transcriptionally shortly after initiation guanylyltransferase (nuclear) transfers G residue to 5 end methyltransferases (nuclear and cytoplasmic) add methyl groups to 5 terminal G and at two 2 ribose positions on the next two nucleotides
pppNpN
mGpppNmpNm
capping involves formation of a 5- 5 triphosphate bond cap function protects 5 end of mRNA (increases mRNA stability) required for initiation of protein synthesis
Polyadenylation cleavage of the primary transcript occurs approximately 10-30 nucleotides 3-ward of the AAUAAA consensus site polyadenylation catalyzed by poly(A) polymerase approximately 200 adenylate residues are added
cleavage
AAUAAA mGpppNmpNm
AAUAAA mGpppNmpNm
A A A
polyadenylation
A A 3
poly(A) is associated with poly(A) binding protein (PBP) function of poly(A) tail is to stabilize mRNA
Chemistry of mRNA splicing two cleavage-ligation reactions transesterification reactions - exchange of one phosphodiester bond for another - not catalyzed by traditional enzymes branch site adenosine forms 2, 5 phosphodiester bond with guanosine at 5 end of intron
intron 1
Pre-mRNA
2OH-A
exon 1
G-p-G-U -
A-G-p-G
exon 2
U-G-5-p-2-A A
Splicing intermediate
exon 1
G-OH O 3
exon 2 A-G-p-G A -
Lariat
U-G-5-p-2-A A
Spliced mRNA
5 exon 1 G-p-G
3 G-A exon 2
Mutations that disrupt splicing bo-thalassemia - no b-chain synthesis b+-thalassemia - some b-chain synthesis
Normal splice pattern:
Exon 1 Intron 1 Donor site: /GU Exon 2 Intron 2 Acceptor site: AG/ Exon 3
Intron 2 acceptor site b mutation: no use of mutant site; use of cryptic splice site in intron 2
Translation of the retained portion of intron 2 results in premature termination of translation due to a stop codon within the intron, 15 codons from the cryptic splice site
Exon 1 Intron 1
Exon 2
Patterns of alternative exon usage one gene can produce several (or numerous) different but related protein species (isoforms)
Cassette
Mutually exclusive
Alternative promoters
The Troponin T (muscle protein) premRNA is alternatively spliced to give rise to 64 different isoforms of the protein Constitutively spliced exons (exons 1-3, 9-15, and 18) Mutually exclusive exons (exons 16 and 17) Alternatively spliced exons (exons 4-8)
Exons 4-8 are spliced in every possible way giving rise to 32 different possibilities Exons 16 and 17, which are mutually exclusive, double the possibilities; hence 64 isoforms
Overview of translation
last step in the flow of genetic information definition of translation requirements for protein synthesis mRNA ribosomes initiation factors elongation and termination factors GTP aminoacyl tRNAs amino acids aminoacyl tRNA synthetases ATP
Cap m7Gppp
5 untranslated region
initiation codon
AUG
UGA
3 untranslated region termination codon
AAUAAA
(AAAA)n 3
poly(A) tail
50S subunit
23S rRNA 5S rRNA 35 proteins
70S ribosome
30S subunit
16S rRNA 21 proteins
eukaryotic ribosome
60S subunit
28S rRNA 5S rRNA 5.8S rRNA 49 proteins
80S ribosome
40S subunit
18S rRNA 33 proteins
N
5 AUG UGA
polysome
subunits dissociate
Transfer RNA tRNA is the adaptor molecule in protein synthesis acceptor stem CCA-3 terminus to which amino acid is coupled carries amino acid on terminal adenosine anticodon stem and anticodon loop
aminoacyl tRNA synthetases are the enzymes that charg 20 amino acids one aminoacyl tRNA synthetase for each amino acid can be several different isoacceptor tRNAs for each am all isoacceptor tRNAs for an amino acid use the same sy
each aminoacyl tRNA synthetase binds amino acid ATP isoacceptor tRNAs
ATP
RO H2N-C-C-OH H - = - =
amino acid
uncharged tRNA 3
PPi
- =
AUG (methionine) is the start codon (also used interna multiple codons for a single amino acid = degeneracy
Tyr
Stop
UGU Cys UGC UGA Stop UGG Trp CGU CGC Arg CGA CGG AGU Ser AGC AGA Arg AGG GGU GGC Gly GGA GGG
His
Gln
Leu
Ile Met
Asn
Lys
Asp
Glu
Val
Codon-anticodon interactions codon-anticodon base-pairing is antiparallel the third position in the codon is frequently degenerate one tRNA can interact with more than one codon (therefore 50 tRNAs) wobble rules C with G or I (inosine) A with U or I 3 5 tRNAmet G with C or U U with A, G, or I I with C, U, or A UAC AUG mRNA
5
3 one tRNAleu can read two of the leucine codons GAU CUA G 5 tRNAleu
wobble base
mRNA 5
Wobble Interactions
Inosine = Cytidine Inosine = Adenosine
Inosine = Uridine
Guanosine = Uridine
Initiation in prokaryotes and eukaryotes initiation can occur at internal AUG codons in prokaryotic mRNA initiation in eukaryotes occurs only at the first AUG codon lac operon in E. coli is transcribed as a polycistronic mRNA with multiple AUG codons
lac I
O
AUG
lac Z
AUG
lac Y
AUG
lac A
AUG
SD
AUG
SD
AUG
5 cap
AUG
initiation can only occur at first AUG codon downstream of the 5 cap
Reading frame
reading frame is determined by the AUG initiation codon every subsequent triplet is read as a codon until reaching a stop codon
...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA... MET.ALA.GLU.TRP.LEU.SER.MET.SER
a frameshift mutation
...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...
the new reading frame results in the wrong amino acid sequence and the formation of a truncated protein
...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA... MET.ALA.ASP.GLY
missense mutations (e.g., AGC Ser to AGA Arg) nonsense mutations (e.g., UGG Trp to UGA Stop) read through, reverse terminator, or sense mutations (e.g., UAA Stop to CAA Gln) as in hemoglobin Constant Spring
silent mutations (e.g., CUA Leu to CUG Leu) do not affect translation
Learning Objectives for Lecture 8: Understand the structure of the ribosome in the context of the translation process Understand the steps in the initiation of protein synthesis Understand the mechanism of peptide bond formation, and that it is RNA catalyzed Understand the processes of elongation and termination Understand how interferon inhibits viral protein synthesis Understand the mechanisms by which antibiotics inhibit protein synthesis how some organisms become resistant to antibiotics Understand how secreted and membrane-bound proteins are synthesized Understand how proteins are glycosylated and what the functions of the carbohydrates are Understand the role of proteolytic processing in protein maturation
and
Ribosome structure
P PP
P P P P P A
Large subunit
5 Small subunit
mRNA
The small subunit finds the 5 cap and scans down the mRNA to the first AUG codon 5 cap AUG mRNA
60S subunit the initiation codon is recognized eIF2 dissociates from the complex the large ribosomal subunit binds
eIF2
M
mRNA
A M
AUG GCC
mRNA
M A aminoacyl tRNA binds the A-site first peptide bond is formed 5 initiation is complete AUG GCC mRNA
peptide bond formation is catalyzed by peptidyl transferase peptidyl transferase is contained within NH2 a sequence of 23S rRNA in the CH3-S-CH2-CH2-CH prokaryotic large ribosomal subunit; therefore, it is probably within O=C the 28S rRNA in eukaryotes NH the energy for peptide bond formation CH3-CH comes from the ATP used in tRNA charging O=C peptide bond formation results in a shift of the nascent peptide from the P-site OH O to the A-site
tRNA
tRNA
Structure shows only RNA in the active site Adenine 2451 carries out acid-base catalysis
Cech (2000) Science 289:878-879 Ban et al. (2000) Science 289:905-920 Nissen et al. (2000) Science 289:920-930
Elongation
P P P P P
UCA GCA GGG UAG
following peptide bond formation the uncharged tRNA dissociates from the P-site the ribosome shifts one codon along the mRNA, moving peptidyl tRNA from the A-site to the P-site; this translocation requires the elongation factor EF2 the next aminoacyl tRNA then binds within the A-site; this tRNA binding requires the elongation factor EF1 energy for elongation is provided by the hydrolysis of two GTPs: one for translocation one for aminoacyl tRNA binding
EF1 EF2
A
P P P P P
UCA GCA GGG UAG
Termination
RF
P P P P P
UCA GCA GGG UAG
when translation reaches the stop codon, a release factor (RF) binds within the A-site, recognizing the stop codon
PPPP PPP P
release factor catalyzes the hydrolysis of the completed polypeptide from the peptidyl tRNA, and the entire complex dissociates
cell makes interferon in response to viral RNA interferon binds to receptors on neighboring ce virus cell cannot and activates the cells itself virus invades protect cell cell synthesizes antiviral proteins virus replicates in response to cell succumbs interferon activation virus invades neighboring cell cell protected from viral infection by antiviral proteins
interferon induces
cytosol
1. translation initiates as usual on a cytosolic mRNA athe signal recognition particle (SRP) consists of protein and RNA (7SL RNA); it binds to the signal peptide, to the ribosome, and to the SRP receptor on the ER membrane bthe signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acids cER = endoplasmic reticulum
4. translation resumes and the nascent polypeptide moves into the ER lumen 5. signal peptidase, which is in the ER lumen, cleaves off the signal peptide
ER lumen cytosol
5
7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes
8. translation continues with the nascent polypeptide emerging into the ER lumen
9. at termination of translation, the completed protein is within the ER and is further processed prior to secretion
ER lumen
cytosol
UGA
Examples of secreted proteins: polypeptide hormones (e.g., insulin) albumin collagen immunoglobulins
Integral membrane proteins are also synthesized by the same mechanisms; they may be considered partially secreted Examples of integral membrane proteins: polypeptide hormone receptors (e.g., insulin receptor) transport proteins ion channels cytoskeletal anchoring proteins (e.g., band 3)
Glycosylation of proteins
most integral membrane proteins and secreted proteins are glycosylated during translation on the ER membrane the protein begins to be glycosylated various oligosaccharide modifications occur in the ER and in the Golgi complex
to hydroxyl group) N-linked (Asn linked) oligosaccharides (linked to amide group) Biosynthesis of N-linked oligosaccharides (first 7 steps) P Dolichol phosphate (polyprenol lipid (2) UDPER lumen (1) UMP, (1) UDP (5) GDP(5) GDP
Cytosol
reorientation
7 steps)
PP P (4)
ER lumen
PP P
(3)
Dolicol-phosphates are the sugar donors in the ER lumen; they are synthesized in the cytosol prior to being translocated to the lumen P Dolicol-P-mannose = P Dolicol-P-glucose =
PP
Cytosol
PP
ER lumen
Asn I X Linkage is to the amide group of an aspa I followed Ser (Thr) by any (X) amino acid (except p followed by serine or threonine
Cytosol
Following synthesis, the protein is transferr to the Golgi complex, where trimming and fu building of the oligosaccharides occurs
Asn
I
X
I
Ser (Thr)
Golgi lumen
Asn
I I
Cytosol
X A complex type oligosaccharide Ser (Thr) fucose = galactose = sialic ac come from nucleotide sugars transloca The type of carbohydrate across the Golgidetermines membrane wh the protein is targeted to the membran
Asn
Proteins containing mannose-6-phospha are targeted to lysoso These proteins include lysosomal hydrolases Phosphate groups are mannose by transfer phosphate from UDP Patients with I-cell (for body) disease have a in the enzyme that tra GlcNAc phosphate to As a result, the hydrola residues in the Golgi be targeted to the lys The resulting deficienc lysosomal hydrolases an accumulation (inc
Proteolytic processing
Processing of insulin (synthesized in the ER of pancreatic b-cells)
Signal peptide
cleavage of signal peptide C by signal Preproinsulin peptidase B-chain Insulin N Further trimming by a S S N I I S S carboxypeptidase B-like A-chain S S C I I N S S C C enzyme removes two C-chain basic residues from by trypsin-like enzyme each of Cleavage the new ends releases the C-peptide The C-chain is C-chain packaged in the secretory
Preproopiomelanocortin
multiple functional polypeptides from a single prec processed in a cell-specific manner 26aa N Signal peptide 48aa 12aa 40aa Proopiomelanocortin 5aa 21aa 14aa C
g-Lipotropin Enkephalin (