THE FLOW OF GENETIC INFORMATION

DNA RNA PROTEIN

DNA

1

2 3

1. REPLICATION (DNA SYNTHESIS)2. TRANSCRIPTION (RNA SYNTHESIS)3. TRANSLATION (PROTEIN SYNTHESIS)

DNA Structure and Chemistry

a). Evidence that DNA is the genetic informationi). DNA transformation – know this termii). Transgenic experiments – know this processiii). Mutation alters phenotype – be able to define

genotype and phenotypeb). Structure of DNA

i). Structure of the bases, nucleosides, and nucleotidesii). Structure of the DNA double helixiii). Complementarity of the DNA strands

c). Chemistry of DNAi). Forces contributing to the stability of the double helixii). Denaturation of DNA

Thymine (T)

Guanine (G) Cytosine (C)

Adenine (A)

Structures of the bases

Purines Pyrimidines

5-Methylcytosine (5mC)

[structure of deoxyadenosine]

Nucleoside

Nucleotide

Nomenclature

Purinesadenine adenosineguanine guanosinehypoxanthine inosine

Pyrimidinesthymine thymidinecytosine cytidine

+ribose uracil uridine

Nucleoside NucleotideBase +deoxyribose +phosphate

• polynucleotide chain• 3’,5’-phosphodiester bond

ii). Structure of the DNA double helix

Structure of the DNApolynucleotide chain

5’

3’

A-T base pair

G-C base pair

Chargaff’s rule: The content of A equals the content of T, and the content of G equals the content of C in double-stranded DNA from any species

Hydrogen bonding of the bases

Double-stranded DNA

Major groove

Minor groove

5’ 3’

5’ 3’3’ 5’

“B” DNA

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)

Stacking interactions

Charge repulsion

Ch

arg

e re

pu

lsio

n

Model of double-stranded DNA showing three base pairs

Denaturation of DNA

Double-stranded DNA

A-T rich regions denature first

Cooperative unwinding of the DNA strands

Extremes in pH or high temperature

Strand separationand formation ofsingle-strandedrandom coils

Electron micrograph of partially melted DNA

• A-T rich regions melt first, followed by G-C rich regions

Double-stranded, G-C rich DNA has not yet melted

A-T rich region of DNAhas melted into asingle-stranded bubble

Hyperchromicity

The absorbance at 260 nm of a DNA solution increases when the double helix is melted into single strands.

260

Ab

sorb

ance

Absorbance maximumfor single-stranded DNA

Absorbancemaximum fordouble-stranded DNA

220 300

100

50

0

7050 90

Temperature oC

Pe

rce

nt

hyp

erc

hro

mic

ity

DNA melting curve

• Tm is the temperature at the midpoint of the transition

Average base composition (G-C content) can bedetermined from the melting temperature of DNA

50

7060 80

Temperature oC

Tm is dependent on the G-C content of the DNA

Pe

rce

nt

hyp

erc

hro

mic

ity

E. coli DNA is 50% G-C

Genomic DNA, Genes, Chromatin

a). Complexity of chromosomal DNAi). DNA reassociationii). Repetitive DNA and Alu sequencesiii). Genome size and complexity of genomic DNA

b). Gene structurei). Introns and exonsii). Properties of the human genome iii). Mutations caused by Alu sequences

c). Chromosome structure - packaging of genomic DNAi). Nucleosomes

ii). Histonesiii). Nucleofilament structureiv). Telomeres, aging, and cancer

DNA reassociation (renaturation)

Double-stranded DNA

Denatured,single-strandedDNA

Slower, rate-limiting,second-order process offinding complementarysequences to nucleatebase-pairing

k2

Faster,zipperingreaction toform longmoleculesof double-strandedDNA

Cot1/2

DNA reassociation kinetics for human genomic DNA

Cot1/2 = 1 / k2 k2 = second-order rate constant Co = DNA concentration (initial) t1/2 = time for half reaction of each

component or fraction

50

100

0

% D

NA

re

ass

oc

iate

d

I I I I I I I I I

log Cot

fast (repeated)

intermediate (repeated)

slow (single-copy)

Kinetic fractions: fast intermediate slow

Cot1/2

Cot1/2

high k2

106 copies per genome ofa “low complexity” sequence

of e.g. 300 base pairs

1 copy per genome ofa “high complexity” sequence

of e.g. 300 x 106 base pairs

low k2

Type of DNA % of Genome Features

Single-copy (unique) ~75% Includes most genes 1

Repetitive Interspersed ~15% Interspersed throughout genome between

and within genes; includes Alu sequences 2

and VNTRs or mini (micro) satellites Satellite (tandem) ~10% Highly repeated, low complexity sequences

usually located in centromeres and telomeres

2 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.

1 Some genes are repeated a few times to thousands-fold and thus would be in the repetitive DNA fraction

50

100

0

I I I I I I I I I

fast ~10%

intermediate ~15%

slow (single-copy) ~75%

Classes of repetitive DNA

Interspersed (dispersed) repeats (e.g., Alu sequences)

TTAGGGTTAGGGTTAGGGTTAGGG

Tandem repeats (e.g., microsatellites)

GCTGAGG GCTGAGGGCTGAGG

viruses

plasmids

bacteria

fungi

plants

algae

insects

mollusks

reptiles

birds

mammals

Genome sizes in nucleotide pairs (base-pairs)

104 108105 106 107 10111010109

The size of the humangenome is ~ 3 X 109 bp;almost all of its complexityis in single-copy DNA.

The human genome is thoughtto contain ~30,000 to 40,000 genes.

bony fish

amphibians

5’ 3’

promoter region

exons (filled and unfilled boxed regions)

introns (between exons)

transcribed region

translated region

mRNA structure

+1

Gene structure

The (exon-intron-exon)n structure of various genes

-globin

HGPRT(HPRT)

total = 1,660 bp; exons = 990 bp

histone

factor VIII

total = 400 bp; exon = 400 bp

total = 42,830 bp; exons = 1263 bp

total = ~186,000 bp; exons = ~9,000 bp

Properties of the human genome

Nuclear genome

• the haploid human genome has ~3 X 109 bp of DNA• single-copy DNA comprises ~75% of the human genome• the human genome contains ~30,000 to 40,000 genes• most genes are single-copy in the haploid genome• genes are composed of from 1 to >75 exons• genes vary in length from <100 to >2,300,000 bp• Alu sequences are present throughout the genome

Mitochondrial genome

• circular genome of ~17,000 bp• contains <40 genes

Familial hypercholesterolemia• autosomal dominant• LDL receptor deficiency

Alu sequences can be “mutagenic”

From Nussbaum, R.L. et al. "Thompson & Thompson Genetics in Medicine," 6th edition (Revised Reprint), Saunders, 2004.

LDL receptor gene

Alu repeats present within introns

Alu repeats in exons

4

4

4

5

5

5 6

6

6

Alu Alu

AluAlu

X

4 6Alu

unequalcrossing over

one product has a deleted exon 5(the other product is not shown)

Chromatin structure

EM of chromatin shows presence ofnucleosomes as “beads on a string”

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 chargedPhosphorylationPoly(ADP) ribosylationMethylationAcetylation

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

Condensation and decondensation of a chromosome in the cell cycle

Telomeres and aging

Metaphase chromosome

centromere

telomere telomere

telomere structure

young

senescent

Telomeres are protective“caps” on chromosomeends consisting of short5-8 bp tandemly repeatedGC-rich DNA sequences,that prevent chromosomesfrom fusing and causingkaryotypic rearrangements.

(TTAGGG)many

(TTAGGG)few

• telomerase (an enzyme) is required to maintain telomere length in germline cells

• most differentiated somatic cells have decreased levels of telomerase and therefore their chromosomes shorten with each cell division

<1 to >12 kb

The mammalian cell cycle

G1

S

G2M

G0

DNA synthesis and histone synthesis

Growth and preparation forcell division

Rapid growth and preparation forDNA synthesis

Quiescent cells

phase

phase

phase

phase

Mitosis

DNA replication is semi-conservative

Parental DNA strands

Daughter DNA strands

Each of the parental strands serves as a template for a daughter strand

origins of DNA replication (every ~150 kb)

replication bubble

daughter chromosomes

fusion of bubbles

bidirectional replication

Origins of DNA replication on mammalian chromosomes

5’3’

3’5’

5’3’

3’5’3’5’

5’3’

Initiation of DNA synthesis at the E. coli origin (ori)

5’3’

3’5’

origin DNA sequence

binding of dnaA proteins

A A A

dnaA proteins coalesce

DNA melting inducedby the dnaA proteinsA

AA

AA

A

AA

AA

A

A B C

dnaB and dnaC proteins bind to the single-stranded DNA

dnaB further unwinds the helix

A

A

A

AA

A B C

dnaB further unwinds the helix and displaces dnaA proteins

GdnaG (primase) binds...

A

A

A

AA

AB C

G...and synthesizes an RNA primer

RNA primer

B C

G

5’ 3’template strand

RNA primer(~5 nucleotides)

Primasome dna B (helicase) dna C dna G (primase)

OH3’ 5’

3’

5’ 3’

RNA primer

newly synthesized DNA

5’

5’

DNA polymerase

Discontinuous synthesis of DNA

3’5’

5’ 3’

3’ 5’

Because DNA is always synthesized in a 5’ to 3’ direction,synthesis of one of the strands...

5’3’ ...has to be discontinuous.

This is the lagging strand.

5’3’

3’5’

5’3’

3’5’

5’ 3’

3’ 5’

5’3’

3’5’

5’3’

leading strand (synthesized continuously)

lagging strand (synthesized discontinuously)

Each replication fork has a leading and a lagging strand

• 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)

replication fork replication fork

RNA primer

5’3’

3’5’

3’5’

direction of leading strand synthesis

direction of lagging strand synthesis

replication fork

5’3’

3’5’

3’5’

Strand separation at the replication fork causes positivesupercoiling of the downstream double helix

• DNA gyrase is a topoisomerase II, which 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)

5’3’ 5’

3’

Movement of the replication fork

Movement of the replication fork

RNA primerOkazaki fragment

RNA primer

5’

3’

RNA primer5’

DNA polymerase III initiates at the primer andelongates DNA up to the next RNA primer

5’

5’3’

5’

newly synthesized DNA (100-1000 bases) (Okazaki fragment)5’

3’

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

pol III

pol I

newly synthesized DNA (Okazaki fragment)5’

3’

5’3’

DNA ligase seals the gap by catalyzing the formationof a 3’, 5’-phosphodiester bond in an ATP-dependent reaction

5’3’

3’5’

Proteins at the replication fork in E. coli

Rep protein (helicase)

Single-strandbinding protein (SSB)

BC

G Primasome

pol I

pol III

pol III

DNA ligase

DNA gyrase - this is a topoisomerase II, whichbreaks and reseals double-stranded DNA to introducenegative supercoils ahead of the fork

Components of the replication apparatus

dnaA binds to origin DNA sequencePrimasome dnaB helicase (unwinds DNA at origin) dnaC binds dnaB dnaG primase (synthesizes RNA primer)DNA gyrase introduces negative supercoils ahead

of the replication forkRep protein helicase (unwinds DNA at fork)SSB binds to single-stranded DNADNA pol III primary replicating polymeraseDNA pol I removes primer and fills gapDNA ligase seals gap by forming 3’, 5’-phosphodiester bond

Properties of DNA polymerases

DNA polymerases of E. coli_

pol I pol II pol III (core)Polymerization: 5’ to 3’ yes yes yesProofreading exonuclease: 3’ to 5’ yes yes yesRepair exonuclease: 5’ to 3’ yes no no

DNA polymerase III is the main replicating enzymeDNA 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

• all DNA polymerases require a primer with a free 3’ OH group• all DNA polymerases catalyze chain growth in a 5’ to 3’ direction• some DNA polymerases have a 3’ to 5’ proofreading activity

Types and rates of mutation

Type Mechanism Frequency________ Genome chromosome 10-2 per cell division mutation missegregation

(e.g., aneuploidy)

Chromosome chromosome 6 X 10-4 per cell division mutation rearrangement

(e.g., translocation)

Gene base pair mutation 10-10 per base pair per mutation (e.g., point mutation, cell division or

or small deletion or 10-5 - 10-6 per locus per insertion generation

Mutation

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 HumanGenome Project as an ongoing project

Types of base pair mutations

CATTCACCTGTACCAGTAAGTGGACATGGT

CATGCACCTGTACCAGTACGTGGACATGGT

CATCCACCTGTACCAGTAGGTGGACATGGT

transition (T-A to C-G) transversion (T-A to G-C)

CATCACCTGTACCAGTAGTGGACATGGT

deletionCATGTCACCTGTACCAGTACAGTGGACATGGT

insertion

base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine

normal sequence

deletions and insertions can involve one or more base pairs

Spontaneous mutations can be caused by tautomers

Tautomeric forms of the DNA bases

Adenine

Cytosine

AMINO IMINO

Guanine

Thymine

KETO ENOL

Tautomeric forms of the DNA bases

Mutation caused by tautomer of cytosine

Cytosine

Cytosine

Guanine

Adenine

• cytosine mispairs with adenine resulting in a transition mutation

Normal tautomeric form

Rare imino tautomeric form

Mutation is perpetuated by replication

• replication of C-G should give daughter strands each with C-G

• tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands

• which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired

C G C G

C G C A

AC T A

Chemical mutagens

Deamination by nitrous acid

N

NH

NH

N

NH2

O

N

NH

NH

NH

NH2

O

O

Attack by oxygen free radicalsleading to oxidative damage

guanine

8-oxyguanine (8-oxyG)

• many different oxidative modifications occur• by smoking, etc.• 8-oxyG causes G to T transversions

• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation• mutation of the MTH1 gene causes increased tumor formation in mice

Ames test for mutagen detection

• named for Bruce Ames• reversion of histidine mutations by test compounds• His- Salmonella typhimurium cannot grow without histidine

• if test compound is mutagenic, reversion to His+ may occur• reversion is correlated with carcinogenicity

Thymine dimer formation by UV light

Summary of DNA lesions

Missing base Acid and heat depurination (~104 purinesper day per cell in humans)

Altered base Ionizing radiation; alkylating agents Incorrect base Spontaneous deaminations

cytosine to uraciladenine to hypoxanthine

Deletion-insertion Intercalating reagents (acridines) Dimer formation UV irradiation Strand breaks Ionizing radiation; chemicals (bleomycin) Interstrand cross-links Psoralen derivatives; mitomycin C Tautomer formation Spontaneous and transient

Mechanisms of Repair

• Mutations that occur during DNA replication are repaired whenpossible by proofreading by the DNA polymerases

• Mutations that are not repaired by proofreading are repairedby mismatch (post-replication) repair followed byexcision repair

• Mutations that occur spontaneously any time are repaired byexcision repair (base excision or nucleotide excision)

Deamination of cytosine can be repaired

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

Deamination of 5-methylcytosine cannot be repaired

cytosine uracil

thymine5’-methyl-cytosine

DNA repair activity

Life

spa

n

1

10

100humanelephant

cow

hamsterratmouseshrew

Correlation between DNA repairactivity in fibroblast cells fromvarious mammalian species andthe life span of the organism

Defects in DNA repair or replicationAll are associated with a high frequency of chromosome

and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemias

• Xeroderma pigmentosum• caused by mutations in genes involved in nucleotide excision repair• associated with a >1000-fold increase of sunlight-induced skin cancer and with other types of cancer such as melanoma

• Ataxia telangiectasia• caused by gene that detects DNA damage• increased risk of X-ray• associated with increased breast cancer in carriers

• Fanconi anemia• caused by a gene involved in DNA repair• increased risk of X-ray and sensitivity to sunlight

• Bloom syndrome• caused by mutations in a a DNA helicase gene• increased risk of X-ray• sensitivity to sunlight

• Cockayne syndrome• caused by a defect in transcription-linked DNA repair• sensitivity to sunlight

• Werner’s syndrome• caused by mutations in a DNA helicase gene• premature aging

3. RNA Structure and Transcription

a). Chemistry of RNAi). Bases found in RNAii). Ribose sugariii). RNA polynucleotide chainiv). Secondary and tertiary structure

b). Characteristics of prokaryotic RNAi). Classes of prokaryotic RNAii). Structure of prokaryotic messenger RNA

c). Transcription initiation in prokaryotesi). Transcriptionii). Promoter structureiii). Prokaryotic RNA polymerase structureiv). Initiation of transcription and the sigma cycle

d). Regulation of the lactose operoni). Function of the lactose operonii). Negative control: Lac repressor and induceriii). Positive control: CAP and cAMP

The major bases found in DNA and RNA

DNA RNA

Adenine Adenine Cytosine Cytosine Guanine Guanine Thymine Uracil (U)

uracil-adenine base pairthymine-adenine base pair

RNA polynucleotide chain

• 2’ -OH makes 3’, 5’ phosphodiester bond unstable

DNA polynucleotide chain

Tertiary structure

Secondary structure

• ribosomal RNA (rRNA)16S (small ribosomal subunit)23S (large ribosomal subunit)5S (large ribosomal subunit)

• transfer RNA (tRNA)• messenger RNA (mRNA)

Structure of prokaryotic messenger RNA

5’

3’

PuPuPuPuPuPuPuPu AUGShine-Dalgarno sequence initiation

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis

Classes of prokaryotic RNA

AAUtermination

translated region

Transcription

RNA polymerase

closed promoter complex

open promoter complex

initiation

elongation

termination

RNA product

Promoter structure in prokaryotes

5’ PuPuPuPuPuPuPuPu AUG

Promoter

+1 +20-7-12-31-36

5’mRNA

mRNA

TTGACAAACTGT

-30 region

TATAATATATTA

-10 region

8479 53 45%82T T G

64AC A

79T

44T

96%T

95A

59A

51A

consensus sequences

-30 -10

transcription start site

Pribnow box

+1[ ]

Prokaryotic RNA polymerase structure

RNA polymerase of bacteria is a multisubunit protein

Subunit Number Role

2 uncertain

(Rifampicin target) 1 forms phosphodiester bonds

’ 1 binds DNA template

1 recognizes promoter and facilitates initiation

’ ’ + holoenzyme core polymerase sigma factor

RNA polymerase holoenzyme (+ 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

• open promoter complex (highly stable)• the holoenzyme has very high affinity for promoter regions because of sigma factor

• sigma can re-bind other core enzymes The sigma cycle

Mechanism of RNA synthesis

• RNA synthesis usually initiated with ATP or GTP (the first nucleotide)• RNA chains are synthesized in a 5’ to 3’ direction

A = T

U = A

A = T

U = A

RNA RNA

lac I P Opromoter - operator

lac repressor

lac Z lac Y lac A

The lactose operon in E. coli

-galactosidase permease acetylase

LACTOSE GLUCOSE + GALACTOSE-galactosidase

•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

• promoter binds CAP and RNA polymerase• operator binds the lac repressor

Regulation of the lactose operon - negative control

lac I P Opromoter - operator

lac repressor

lac I P lac Z lac Y lac A

• the repressor tetramer binds to the operator and prevents RNA polymerase from binding to the promoter

lac Z lac Y lac A

NO TRANSCRIPTION

RNA pol • RNA polymerase is blocked from the promoter

NO TRANSCRIPTION

• when lactose becomes available, it is taken up by the cell• allolactose (an intermediate in the hydrolysis of lactose) is produced• one molecule of allolactose binds to each of the repressor subunits• binding of allolactose results in a conformational change in the repressor• the conformational change results in decreased affinity of the repressor for the operator and dissociation of the repressor from the DNA

Alleviation of negative control - action of the inducer of the lac operon

allolactose

lac I P lac Z lac Y lac A

lac I P lac Z lac Y lac A

• IPTG (isopropyl thiogalactoside) is also used as a (non-physiological) inducer

lac I P lac Z lac Y lac A

NO TRANSCRIPTION

RNA pol

O

• repressor (with bound allolactose) dissociates from the operator• negative control (repression) is alleviated, however...

• RNA polymerase cannot form a stable complex with the promoter

lac I P O lac Z lac Y lac A

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 inactive CAPcAMP

+

NO TRANSCRIPTION 2 also termed catabolite activator protein

lac I

lac repressor

lac Z lac Y lac A

-galactosidase permease acetylase

RNA pol

TRANSCRIPTION AND TRANSLATION OCCUR

inactive repressor

Activation of lac operon transcription

• 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 processingi). Cappingii). Cleavage and polyadenylationiii). Splicing

b). Chemistry of mRNA splicingc). Spliceosome assembly and splice site recognition

i). Donor and acceptor splice sitesii). Small nuclear RNAs

d). Mutations that disrupt splicinge). 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

cap

cap

cap poly(A)

cap poly(A)

Transcription of pre-mRNA and capping at the 5’ end

Cleavage of the 3’ end and polyadenylation

Splicing to remove intron sequences

Transport of mature mRNA to the cytoplasm

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 onthe next two nucleotides

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

pppNpN

mGpppNmpNm

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

• poly(A) is associated with poly(A) binding protein (PBP)• function of poly(A) tail is to stabilize mRNA

mGpppNmpNmAAUAAA

mGpppNmpNmAAUAAA AA

A

A

AA

3’

cleavage

polyadenylation

Chemistry of mRNA splicing• two cleavage-ligation reactions• transesterification reactions - exchange of one

phosphodiester bond for another - not catalyzed bytraditional enzymes• branch site adenosine forms 2’, 5’ phosphodiester bond

with guanosine at 5’ end of intron

G-p-G-U A-G-p-G

2’OH-A

-5’ 3’

intron 1

exon 1 exon 2

Pre-mRNA

First clevage-ligation (transesterification) reaction

branch site adenosine

G-OH 3’ A-G-p-G

U-G-5’-p-2’-A

5’ 3’A

A

O -

G-p-G5’ 3’

U-G-5’-p-2’-AA

3’ G-A

Splicingintermediate

Lariat

exon 1

exon 1

exon 2

exon 2

intron 1

intron 1

Second clevage-ligation reaction

Spliced mRNA

• ligation of exons releases lariat RNA (intron)

Mutations that disrupt splicing• o-thalassemia - no -chain synthesis• +-thalassemia - some -chain synthesis

Normal splice pattern:

Exon 1 Exon 2 Exon 3Intron 1 Intron 2

Donor site: /GU Acceptor site: AG/

Intron 2 acceptor site mutation: no use of mutant site; use of cryptic splice site in intron 2

Exon 1 Exon 2Intron 1

mutant site: GG/

Intron 2 cryptic acceptor site: UUUCUUUCAG/G

Translation of the retained portion of intron 2 results in premature termination of translation due to a stop codon within the intron, 15 codons fromthe cryptic splice site

Patterns of alternative exon usage• one gene can produce several (or numerous) different

but related protein species (isoforms)

Cassette

Mutually exclusive

Internal acceptor site

Alternative promoters

The Troponin T (muscle protein) pre-mRNA

is alternatively spliced to give rise to64 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 possibilitiesExons 16 and 17, which are mutually

exclusive,double the possibilities; hence 64 isoforms

7. Protein Synthesis and the Genetic Code

a). Overview of translationi). Requirements for protein synthesisii). messenger RNAiii). Ribosomes and polysomesiv). Polarity of protein synthesis

b). Transfer RNAi). tRNA as an adaptorii). Amino acid activationiii). Aminoacyl tRNA synthetasesiv). “Charged” tRNA

c). The genetic codei). Codon-anticodon interactionsii). Initiation codon in prokaryotes vs. eukaryotesiii). Reading frame

d). Mutations affecting translationi). Frameshift mutationsii). Missense and nonsense mutations

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

Messenger RNA (mRNA)

m7Gppp

Cap

5’5’ untranslated region

AUG

initiation codon

translated (coding) region

(AAAA)n

poly(A) tail

3’ untranslated region

UGAtermination codon

3’AAUAAA

Ribosomes• prokaryotic ribosome

• eukaryotic ribosome

70S ribosome

80S ribosome

50S subunit 23S rRNA 5S rRNA 35 proteins

60S subunit 28S rRNA 5S rRNA 5.8S rRNA 49 proteins

30S subunit 16S rRNA 21 proteins

40S subunit 18S rRNA 33 proteins

Polysomes• direction of translation is 5’ to 3’ along the mRNA

• direction of protein synthesis is N terminus to C terminus

UGA5’

large ribosomal subunit

small ribosomal subunit

AUG

polysome

nascentpolypeptide

NN

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

Amino acid activation and aminoacyl tRNA synthetases

• aminoacyl tRNA synthetases are the enzymes that “charge” the tRNAs• 20 amino acids• one aminoacyl tRNA synthetase for each amino acid• can be several different “isoacceptor” tRNAs for each amino acid• all isoacceptor tRNAs for an amino acid use the same synthetase

• each aminoacyl tRNA synthetase binds• amino acid• ATP• isoacceptor tRNAs

H2N-C-C-OHH

R--

O=ATP

H2N-C-C-O-P-O-ribose-adenineH

R--

O=

amino acid

adenylated (activated)amino acid

PPi

uncharged tRNA

H2N-C-C-OH

R--

O=

aminoacyl(charged)

tRNA

AMP

3’

Amino acid activationand

tRNA charging

The genetic code

• consists of 64 triplet codons (A, G, C, U) 43 = 64

• all codons are used in protein synthesis• 20 amino acids• 3 termination (stop) codons: UAA, UAG, UGA

• AUG (methionine) is the start codon (also used internally)

• multiple codons for a single amino acid = degeneracy

• 5 amino acids are specified by the first two nucleotides only

• 3 additional amino acids (Arg, Leu, and Ser) are specified bysix different codons

The Genetic Code

UUUUUCUUAUUG

CUUCUCCUACUG

AUUAUCAUAAUG

GUUGUCGUAGUG

UCUUCCUCAUCG

CCUCCCCCACCG

ACUACCACAACG

GCUGCCGCAGCG

UAUUACUAAUAG

CAUCACCAACAG

AAUAACAAAAAG

GAUGACGAAGAG

UGUUGCUGAUGG

CGUCGCCGACGG

AGUAGCAGAAGG

GGUGGCGGAGGG

Phe

Leu

Leu

Val

Ile

Met

Ser

Pro

Thr

Ala

Tyr

Stop

His

Gln

Asn

Lys

Asp

Glu

Cys

Arg

Ser

Arg

Gly

StopTrp

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• G with C or U• U with A, G, or I• I with C, U, or A

5’ 3’

A U G

U A C

3’ 5’ tRNAmet

mRNA

5’ 3’

C U A G

G A U

3’ 5’ tRNAleu

mRNA

wobble base

• one tRNAleu can read two of the leucine codons

Inosine = Cytidine Inosine = Adenosine

Inosine = Uridine Guanosine = Uridine

Wobble Interactions

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

•

• eukaryotic mRNA

P O lac Z lac Y lac AAUG AUG AUG

AUGSD AUGSDAUG

initiation codon with Shine-Dalgarno site

initiation codon with Shine-Dalgarno site

internal Met codondoes not have

Shine-Dalgarno site

5’

5’ cap AUG

initiation can only occur atfirst AUG codon downstream of the 5’ cap

AUG

internal (downstream) Met codon cannot serve as an initiation site

AUG

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 andthe formation of a truncated protein

...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA... MET.ALA.ASP.GLY

Mutations affecting translation• hemoglobin Wayne (3’ terminal frameshift mutation)

Normal -globin .ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA.THR.SER.LYS.TYR.ARG

Wayne -globin .ACG.UCA.AAU.ACC.GUU.AAG.CUG.GAG.CCU.CGG.UAG.THR.SER.ASN.THR.VAL.LYS.LEU.GLU.PRO.ARG

mutated region

• 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

8. Protein Synthesis and Protein Processing

a). Ribosome structureb). Protein synthesis

i). Initiation of protein synthesisii). Peptide bond formation; peptidyl transferaseiii). Elongation and terminationiv). Inhibitors of protein synthesis

Antiviral action of interferonInduction of 2-5A synthaseInduction of eIF2 kinase

Antibioticsc). Protein processing

i). Synthesis of secreted and integral membrane proteinsii). Glycosylation and protein targetingiii). Proteolytic processing

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 and 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

Ribosome structure

A

P P PPPPPP

P-sitepeptidyl tRNA site

A-siteaminoacyl tRNA site

mRNA5’

Small subunit

Large subunit

Ribosome with bound tRNAs and mRNA

mRNA5’ cap

40S subunit

M

eIF2

AUG

Initiator tRNA bound to thesmall ribosomal subunit with the

eukaryotic initiation factor-2 (eIF2)

Initiation of protein synthesis: mRNA binding

The small subunit finds the 5’ cap andscans down the mRNA to the first AUG codon

mRNA5’

40S subunit

M

eIF2

AUG

• the initiation codon is recognized• eIF2 dissociates from the complex• the large ribosomal subunit binds

60S subunit

mRNA5’

M

AUG

• aminoacyl tRNA binds the A-site

• first peptide bond is formed

• initiation is complete

GCC

A

mRNA5’

M

AUG GCC

A

C

NH2

CH3-S-CH2-CH2-CH O=C

Peptide bond formation

• peptide bond formation iscatalyzed by peptidyl transferase

• peptidyl transferase is contained withina sequence of 23S rRNA in theprokaryotic large ribosomal subunit;therefore, it is probably withinthe 28S rRNA in eukaryotes

• the energy for peptide bond formationcomes from the ATP used in tRNA charging

• peptide bond formation results in a shiftof the nascent peptide from the P-siteto the A-site

NH2

CH3-S-CH2-CH2-CH O=C O

tRNA

NH2

CH3-CH O=C O

tRNA

N

P-site A-site

OH

tRNA

NHCH3-CH O=C O

tRNA

Cech (2000) Science 289:878-879Ban et al. (2000) Science 289:905-920Nissen et al. (2000) Science 289:920-930

Large ribosomal subunit

Protein (purple)lies on the surface

23S RNA (orange and white) makesup the core of the subunit

• Structure shows only RNAin the active site

• Adenine 2451 carries outacid-base catalysis

P

UCA

PPPP

P

UCA GCA GGG UAG

A

PPP

P

Elongation

GCA GGG UAG

• following peptide bond formationthe uncharged tRNA dissociatesfrom the P-site

• the ribosome shifts one codon alongthe mRNA, moving peptidyl tRNAfrom the A-site to the P-site; thistranslocation requires theelongation factor EF2

• the next aminoacyl tRNA thenbinds within the A-site; this tRNAbinding requires the elongationfactor EF1

• energy for elongation is provided bythe hydrolysis of two GTPs:• one for translocation• one for aminoacyl tRNA binding

EF1

EF2

P

UCA GCA GGG UAG

PPP

P

Termination

• when translation reaches the stopcodon, a release factor (RF) bindswithin the A-site, recognizing thestop codon

• release factor catalyzes the hydrolysisof the completed polypeptide fromthe peptidyl tRNA, and the entirecomplex dissociates

RF

P

UCA GCA GGG UAG

PPPPP

PP

Induction and action of interferon

virus

virus invades cell

cell makes interferonin response to viral RNA

cell cannotprotect itself

virus replicatescell succumbs

interferon binds toreceptors on neighboring cells

and activates the cells

cell synthesizesantiviral proteinsin response to

interferon activationvirus invades neighboring cell

cell protected from viralinfection by antiviral proteins

Functions of two antiviral proteins

interferoninduces

ATPviral dsRNA

2-5A synthaseoligo 2-5 adenylate (2-5A)

[-A-2’-p-5’-A-2’-p-5’A-] N

eIF2viral dsRNA

eIF2 kinaseeIF2

P

active inactive:viral protein synthesis cannot initiate

inactiveendonuclease

activeendonuclease:

viral mRNA degraded

Inhibitors of protein synthesis

Inhibitor Process Affected Site of Action Kasugamycin initiator tRNA binding 30S subunitStreptomycin initiation, elongation 30S subunitTetracycline aminoacyl tRNA binding A-siteErythromycin peptidyl transferase 50S subunitLincomycin peptidyl transferase 50S subunitClindamycin peptidyl transferase 50S subunit Chloramphenicol peptidyl transferase 50S subunit

Staphylococcus resistance to erythromycin

• certain strains of Staphylococcus can carry a plasmid that encodesan RNA methylase

• this RNA methylase converts a single adenosine residue in 23S rRNAto N6-dimethyladenosine

• this is the site of action of erythromycin, lincomycin, and clindamycin• N6-dimethyladenosine blocks the action of these antibiotics• the organism that produces erythromycin has its own RNA methylase

and thus is resistent to the antibiotic it makes

Protein maturation: modification, secretion, targeting

5’ AUG

polysome for secreted protein

2. the signal recognition particlea (SRP)

binds the signal peptideb and halts translation

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 membranebthe signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acidscER = endoplasmic reticulum

ER lumen c

cytosol

3. the SRP docks with the SRP receptor on the cytosolic side of the ER membrane and positions the signal peptide for insertion through a pore

SRP SRP receptor

Translation of a secreted protein

5’

ER lumen

cytosol

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

7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes

6. the SRP is released and is recycled

5’

ER lumen

cytosol

UGA

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

completed protein is processed andsecreted

• 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

• O-linked (Ser, Thr linked) oligosaccharides (linked to hydroxyl group)• N-linked (Asn linked) oligosaccharides (linked to amide group)

Biosynthesis of N-linked oligosaccharides (first 7 steps)

ER lumen

Cytosol

P

(1) UMP, (1) UDP

Dolichol phosphate (polyprenol lipid carrier)

N-acetylglucosamine (GlcNAc) =

Mannose =

(2) UDP-

PP(5) GDP-

(5) GDP

PPreorientation

Monosaccharides are transferredby specific glycosyltransferases

from nucleotide sugars

PP ER lumen

Dolicol-phosphates are thesugar donors in the ER lumen;

they are synthesized in the cytosolprior to being translocated to the lumen

Cytosol

PP

PP

(4)

(3)Dolicol-P-mannose =

Dolicol-P-glucose =

P

PP

P

Biosynthesis of N-linked oligosaccharides (second 7 steps)

ER lumen

Cytosol

PP

Linkage is to the amide group of an asparaginefollowed by any (X) amino acid (except proline)

followed by serine or threonine

Transfer of oligosaccharide chainto the growing polypeptide

AsnIXI

Ser (Thr)

Following synthesis, the protein is transferredto the Golgi complex, where trimming and further

building of the oligosaccharides occurs

Transfer of oligosaccharide to protein

AsnI

XI

Ser (Thr)

AsnI

XI

Ser (Thr)

Trimming by glycosidases;Building by glycosyltransferases

A complex type oligosaccharide

fucose = galactose = sialic acid =come from nucleotide sugars translocated

across the Golgi membrane

Golgi lumen

CytosolThe type of carbohydrate determines whether

the protein is targeted to the membrane,to a vesicle, or is secreted

= common core structure

Formation of complex type oligosaccharides

Targeting of proteins to lysosomes (I-cell disease)

Asn

Asn

UDP-

P

P

Asn

P

P

• Proteins containing mannose-6-phosphate are targeted to lysosomes

• Patients with I-cell (for inclusion body) disease have a deficiency in the enzyme that transfers GlcNAc phosphate to mannose residues in the Golgi

• Phosphate groups are added to mannose by transfer of GlcNAc phosphate from UDP-GlcNAc

• The resulting deficiency in lysosomal hydrolases results in an accumulation (inclusions) of material in the lysosomes

• These proteins include the lysosomal hydrolases

• As a result, the hydrolases cannot be targeted to the lysosomes

Proteolytic processing

Processing of insulin (synthesized in the ER of pancreatic -cells)

N

CPreproinsulin

cleavage ofsignal peptideby signalpeptidase

Signal peptide

C

SI

S

SI

S

N

Proinsulin

C

SI

S

SI

S

N

C-chain

Cleavage by trypsin-like enzymesreleases the C-peptide

C

SI

S

SI

S

NInsulin

CN

Disulfide bondformation

Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends

C-chain The C-chain is packaged in the secretoryvesicle and is secreted along with active insulin

B-chain

A-chain

Preproopiomelanocortin

• multiple functional polypeptides from a single precursor• processed in a cell-specific manner

26aa 48aa 12aa 40aa 14aa 21aa 40aa 18aa 26aaN C

Signalpeptide

Proopiomelanocortin

Corticotropin(ACTH)

-MSH -Lipotropin

-MSH -MSHEndorphin

-LipotropinEnkephalin (5aa)

31aa

5aa