Part 11 lecture (eukaryotic nucleus)

8/7/2019 Part 11 lecture (eukaryotic nucleus)

1/29

EUKARYOTIC NUCLEUS

Nucleusresponsible for the storage and utilization of genetic

informationStructure:

1. nuclear envelope

>the most important feature that distinguishes eukaryotesfrom prokaryotes>serve as a barrier that prevents ions, solutes, andmacromolecules from passing freely between the nucleusand cytoplasm>consists of 2 cellular membrane:a. outer membrane

- generally studded with ribosomes and continuous withthe membrane of the RER

b. inner membrane- bound by integral proteins to a thin filamentous

meshwork called nuclear lamina


2/29

> contains several thousand nuclear pores, which plays arole in nucleocytoplasmic exchange

2. Chromosomes> threadlike strands that are composed of the nuclear DNAof eukaryotic cells and are the carriers of geneticinformation

3. Nucleoli> irregularly shaped electron-dense structures thatfunction in the synthesis of ribosomal RNA

4. Nucleoplasm> the fluid substance in which the solutes of the nucleusare dissolved

5. Nuclear matrix> protein containing fibrillar network

Genomecollective term for the entire genetic information in every

nucleated eukaryotic cell

I. Physical and Structural Organization of the GenomeA. Deoxyribonucleic acid (DNA)

Chain of nucleotides


3/29

Nucleotides

Nucleoside Phosphate backbone

Nitrogenous base Sugar

purine pyrimidine 2-deoxy-D-ribose D-ribose(DNA) (RNA)

adenine guanine thymine cytosine(DNA)

uracil (for RNA)

Double right hand helix> proposed by Watson and Crick> makes one complete turn every 10 base pairs (34 A 0 or 34nm)> spaces between adjacent turns of the helix form 2 groovesof different width, a wider major groove and a more narrowminor groove


4/29

Double-strand runs anti-parallel (one strand is 5 to 3 whilethe other strand is (3 to 5)

Very stable, due to very precise base-stacking and precise

hydrgogen bonding. Hydrogen bonding follows Chargaffsprinciple: G C and A T or (A + G) / (T + C) = 1

Each nucleotide is linked to one another by phosphodiester bond

Different types of structure of the DNA double helix:1. B-DNA 2. A-DNA 3. Z-DNA

a. Helix Right-handed Right-handed Left-handedb. # of basepairs per 360 0

turn

10 11 12

c. Height of each 360 0 turn

34 A 0 > 34 A 0 < 34 A 0

d. Breadth 20 A 0 < 20 A 0 > 20 A 0

DNA Supercoilingthe formation of additional coils in DNA due to twisting forces2 types:a. Negative supercoiling

> an underwound DNA molecule> have fewer right-hand turns than expected from thenumber of their base pairs

b. Positive supercoiling> an overwound DNA molecule> have more right-hand turns than expected from thenumber of their base pairs

DNA denaturation/DNA melting


5/29

ability of the DNA to separate into its two component strandsbrought about by high temperature (e.g. 100 0 C)

DNA renaturation/ DNA reannealingability of the complementary single-stranded DNA to formback the double helix

Types of DNA sequences:1. Highly repeated DNA sequencesa. satellite DNAsb. minisatellite DNAsc. microsatellite DNAs

2. Moderately Repeated DNA sequencesa. Repeated DNA sequences with coding functionsb. Repeated DNA sequences that lack coding functions

3. Non-repeated DNA sequences

Pseudogenes

sequences that are clearly homologous to functional genes,but have accumulated mutations that render themnonfunctional

TansposonsDNA segments capable of moving from one place in thegenome to another can cause mutation

B. ChromatinDNA molecules with associated histones and smaller amounts proteinsconsist of repeating units called nucleosomes


6/29

Histonesare heterogenous group of closely related arginine and lysinerich basic proteins (Arg and Lys help histones to bind tightlyto the negatively charged sugar phosphate backbone of DNA)provide for the compaction of chromatin

* Prokaryotic DNA is naked because it is not associated withhistones.

Types of Chomatin:1. Euchromatin

chromatin that is not highly compacted and maybetranscriptionally active


7/29

2. Heterochromatinchromatin that is highly compacted and is usuallytranscriptionally inactive2 types:a. Constitutive heterochromatin

> chromatin that remains in the compacted state in allcells at all times, thus, represents DNA that is permanentlysilenced

b. Facultative heterochromatin> chromatin that has been specifically inactivated during

certain phases of an organisms life

C. Chromosomesstructures made up of 2 coiled filaments which are productsof DNA replication during the S phasemade up of 2 chromatids (sister chromatids)Structure:a. centromere

> join the 2 sister chromatids> a boundary that separate the 2 arms> serve as handles, which allow mitotic spindles to attachto the chromosome during cell division

b. p short armc. q long armd. telomeres

> are hexameric DNA repeats (TTAGGG) found at the endsof chromosomes that serve to protect the chromosomesfrom degradation


8/29

Classification of chromosomes based on centromere location:

1. metacentric chromosomecentromere is centrally located

2. submetacentric chromosomecentromere is between the end and middle part

3. acrocentric chromosomecentromere is near the end

4. telocentric chromosomecentromere is at the end

Karyotypea photographic representation of all the chromosomes withina cellreveals how many chromosomes are found within an activelydividing somatic cell

Ploidy the number of chromosome copies within a cell1. Somatic cells

diploid number (46 or 23 pairs)


9/29

each nucleus has 2 copies of each chromosome (homologouschromosome), one from each paent

2. Germ cellshaplod number (22 autosomes and 1 sex chromosome)

Changes in chromosome number:1. Aneuploidy 1 extra or 1 less chromosome

Turner syndrome> inheriting 1 X chromosome with no corresponding X or Ychromosome

2. Polyploidy 3 or more of each type of chromosome

Klinefelter syndrome> inheriting 2 X chromosome and 1 Y chromosome

Downs syndrome (Trisomy 21)> 3 chromosome 21

Chromosomal aberrations and Human Disorders1. Inversions

a chromosome breaks in 2 places, and the segment betweenthe breaks become resealed into the chromosome in reverseorientation

2. Tanslocations

all or a piece of one chromosome becomes attached toanother chromosome

Philadelphia chromosome> a piece of chromosome 22 is taslocated to

chromosome 9> found in certain forms of leukemia


10/29

3. Deletionsa portion of chromosome is missing

Cri-du-chat syndrome> a portion of chromosome 5 is missing

4. Duplicationsa portion of chromosome is repeated

partial trisomy> a condition in which a number of genes are present in 3copies rather than 2 copies

II. Functional Organization of the Genome

In the 1860s, Gregor Mendel mated pea plants havingdifferent inheritable characteristic (height, seed color, seedshape, flower color, flower position, pod color, and pod shape) anddetermined the pattern by which these characteristics weretransmitted to offspring.

Genesbasic unit of inheritance that governs the character of aparticular traitin molecular terms, a segment of DNA containing theinformation for a single polypeptide or RNA moleculethere are 2 copies that control the development of each trait

Genotype particular genes an individual carriesPhenotype individuals observable traits

allelesalternate forms of agenecould be homozygous or heterozygous, dominant or recessive


11/29

homozygous dominant has a pair of dominant alleleshomozygous recessive has a pair of recessive allelesheterozygous has a pair of non-identical alleles

Mendels law of inheritance:1. Law of independent segregation

Even though the pair of alleles that governed a trait remainedtogether throughout life of an individual, they becomeseparated from one another during the formation of gametes

2. Law of independent assortmentThe segregation of the pair of alleles for one trait had no

effect on the segregation of alleles for another traitdoes not apply to all gene combination

Incomplete dominanceone allele of a pair is not fully dominant over its partner, so aheterozygous phenotype somewhere in between the 2homozygous phenotypes emergesEx: Red & White = Pink

CodominanceA pair of nonidentical alleles specify two phenotypes, whichare both expressed at the same time in heterozygotesEx: ABO blood types

PleiotrophyExpression of the alleles at just a single location on a

chromosome may have positive or negative effects on two or more traitsEx: Sickle cell anemia (HbS instead of HbA0


12/29

Inheritance patterns:1. Autosomal dominant

a pattern of inheritance in which the transmission of adominant allele on an autosome causes a trait to beexpressed

A aA AA (expressed) Aa (expressed)a Aa (expressed) aa (not expressed)

2. Autosomal recessivea pattern of inheritance in which the transmission of arecessive allele on both autosomes causes a trait to be

expressedA a

A AA (normal) Aa (carrier)a Aa (carrier) aa (affected)

3. X-linked recessivea mode of inheritance in which a mutation in a gene on the Xchromosome causes the phenotype to be expressed in males

(who are necessarily hemizygous for the gene mutationbecause they have only one X chromosome)

X YX XX (normal) XY (normal)X r XX r (carrier) X r Y(affected)

Non-Mendelian Inheritance:1. Maternal effect

Genotype of the mother directly determines the phenotypictraits of her offspring

2. Epigenetic inheritanceInheritance that is not encoded in the DNA sequence


13/29

The Human Genome Project (HGP)Started in 1990 and completed in 2003Project goals were to:a. Identify all the approximately 20,000-25,000 genes in

human DNAb. Determine the sequences of the 3 billion chemical base

pairs that make up the human DNAc. Store the information in databases (GenBank)d. Improve tools for data analysise. Transfer related technologies to the private sector f. Address the ethical, legal and social issues that may arise

from the project

CENTRAL DOGMA OF MOLECULAR BIOLOGY

ReplicationDuplication of the genetic material

TranscriptionFormation of a complementary RNA from a DNA template

Translation

Synthesis of proteins using the information encoded by anmRNA


14/29

DNA REPLICATION

Characteristics:1. Semiconservative with respect to parental strand

When DNA is replicated during the process of cell division (Sphase), each daughter duplex contains one strand from theparent structure

2. Bidirectional with multiple origins of replication. (Single originonly in the case of prokaryotes)

OriC specific site on E. coli chromosome where replicationbeginsORC(Origin Recognition Complex)

specific site on eukaryotic chromosome where replicationbegins


15/29

3. Primed by short stretches of RNA called primeraccomplished by primase, a type of RNA polymerase

4. Semidiscontinuous with respect to the synthesis of new DNAsynthesis is always in the 5 to 3 directionsynthesis is continuous in one strand (leading strand) whilediscontinuous in another strand (lagging strand)

Okazaki fragmentssmall segments of DNA that are rapidly linked to longer pieces to form the lagging strand

Replication forkthe points at which the pair of replicated segments cometogether and join the nonreplicated segmentscorresponds to a site where:1. the parental double helix is undergoing strand separation2. nucleotides are being incorporated into the newly

synthesized complementary strands

Enzymes Involved in DNA synthesis:1. DNA helicase

Responsible for unwinding the DNA double helix

2. Single-stranded binding protein (ssBP)Prevent premature annealing of the single-stranded DNA todouble stranded DNA

3. DNA Topoisomerase/ DNA gyraseIt relaxes the supercoiling of the DNA, thus helping in theunwinding of the helixPrevents overwinding of the DNA2 types:a. Topoisomerase I

> catalyzes breaks in only one DNA strand


16/29

b. Topoisomerase II> catalyzes breaks in both strands of the DNA

4. Primase initiate synthesis of the primer

5. DNA polymeraseMajor enzyme used in replicating DNAExtend an existing nucleic acid strand in 5 to 3 directionHave 3 to 5 exonuclease activity that allows them toremove nucleotides that are not part of the double helix5 to 3 exonuclease activity is important for removingprimers

Can also perform excision and repair of damaged DNA

Properties of Eukaryotic DNA polymerasePolymerase Location Nucleus Nucleus Mitochondri

aNucleus Nucleus

Replication Yes No Yes No YesRepair No Yes No Yes No

AssociatedFunctions:5-3polymerase

Yes Yes Yes Yes Yes

3-5exonuclease

No No Yes Yes Yes

5-3exonuclease

No No No No No

6. LigaseCatalyzes the sealing of nicks (breaks) remaining in theDNA after DNA polymerase fills the gap left by primase


17/29

Steps in DNA synthesis:1. Recognition of origin of replication by an initiator protein2. Unwinding of the DNA helix3. Formation of the RNA primer 4. Elongation of the new DNA strand5. Termination of the RNA primer 6. Covalent bonding of Okazaki fragments by DNA ligase7. Cutting of the replicated portion by DNA Topoisomerase

DNA damageCan result from both endogenous causes and exogenouscauses such as ionizing radiation, hydrocarbons, oxidative

free radicals and chemotherapeutic agents

DNA repair systems:1. Mismatch repair

Corrects mismatches of base pairing usually due tomistakes made by DNA polymerase during replication


18/29

2. Base excision repair Required to correct the spontaneous depurination andspontaneous deaminationDeamination of cytosine causes it to be converted to uracil

3. Nucleotide excision repair Remove UV light-induced DNA damage as well as DNAdamage from environmental chemicals which formpyrimidine-pyrimidine dimmers

4. Double-stranded DNA repair

Used to repair damage from ionizing radiation, oxidative freeradicals, or chemotherapeutic agents which causes DNAstrands to be severed2 types:a. Homologous recombination

> type of repair which takes advantage of sequenceinformation available from the unaffected homologouschromosome for proper repair of breaks

b. Non-homologous end joining> permits the joining of ends even if there is no sequencesimilarity between them

Xeroderma pigmentosumAn autosomal recessive genetic disorder of DNA repair inwhich patients carry mutations in the nucleotide repair

enzymes (defective repair of thymine dimers)Affected individuals are prone to develop multiple skincancers


19/29

TRANSCRIPTION

A process by which an RNA molecule is polymerized on aDNA template with the aid of various proteinsProduces all types of RNAs:1. rRNA/ ribosomal RNA

transcribed by RNA polymerase I80% of the total RNA in the cellAssociate with proteins to form ribosomes, which areimportant during protein synthesis as they containpeptidyl transferase activity

2. tRNA/ transfer RNAtranscribed by RNA polymerase IIsmallest of all the RNAsclover-leaf structureFunctions in protein synthesis:a. Carry the appropriate amino acidb. Provide a mechanism by which nucleotide

information can be translated to amino acidinformation through its anticodon

3. mRNA/ messenger RNAtranscribed by RNA polymerase IIlinear with hairpin loop structurecarries genetic information from DNA to cytosol for translation

4. Other types of RNA:a. Small nuclear RNAs (snRNAs)b. Small nucleolar RNAs (snoRNAs)c. Small interfering RNAs (siRNAs)d. micro RNAs (miRNAs)


20/29

Gene structure:

1. Exons coding region2. Introns noncoding region3. upstream sequences

noncoding regions at the 5 end to the 1 st exon4. Downstream sequences

found at the 3 end5. Promoters

are DNA conserved sequences that select or determine thestart site of RNA synthesisincludes:a. TATA box

Contains sequences of conserved nucleotides thatsignals start of transcriptionDirects RNA polymerase II to the correct site

b. CAAT box & GC boxSpecifies the frequency of initiating transcription


21/29

Steps in RNA transcription:1. Initiation

Binding of RNA polymerase to the promoter region

2. ElongationComplementary base pairing occur ( C G; A U)

3. TerminationA termination code in the DNA indicates where transcriptionwill stop

Pre-mRNA/ heterogenous nuclear RNA (hnRNA)

Large group of RNA molecules that found only in the nucleusand is usually altered by splicing and other modificationsbefore it exits the nucleusContains segments of transcribed introns


22/29

RNA processing reactions:1. Addition of a 5 cap

The 5 end of RNA is capped by a methyl guanosine residue,which protects it from degradation by 5 exonucleases duringelongation of the RNA chainAlso helps the transcript bind to the ribosome during proteinsynthesis

2. Addition of a poly (A) tail at the 3 end

3. Intron removal

Splice site sequences, which indicate the beginning (GU) andending (AG) of each intron, are found within the primary RNAtranscript

SpliceosomeSplice or join exons together to form the mature RNA

TRANSLATIONCodon

A sequence of three nucleotides in mRNA that functions intranslationStart codon: AUG (codes for methionine)Stop codon: UAA, UAG, UGA

Genetic code

Manner in which the nucleotide sequences of DNA encodethe information for making protein productsEach codon specifies a particular amino acid or the end of translationCharacteristics of the genetic code:1. Specificity


23/29

A particular codon always codes for the same aminoacid

2. UniversalitySpecificity has been conserved from very early stagesof evolution, with only slight differences in the manner in which the code is translated (* In the mitochondria,UGA codes for trp)

3. Redundancy/DegeneracyA given amino acid may have more than 1 tripletcoding for it

4. Nonoverlapping and commalessThe code is read from a fixed starting point as acontinuous sequence of bases, taken 3 at a time


24/29

Components required for translation:1. Amino acids

If one amino acid is missing, translation stops at thecodon specifying that amino acid

2. mRNA serves as template for synthesis of the polypeptide3. Aminoacyl-tRNA synthetase

Catalyzes a 2-step reaction that results in the covalentattachment of the carboxyl group of an amino acid to the3 end of its corresponding tRNA

4. Protein factors for initiation, elongation and termination5. ATP and GTP as source of energy


25/29

6. Ribosomes which are functionally competent2 subunits:a. Larger subunit

> catalyzes the formation of the peptide bonds thatlink amino acid residues in a protein

b. Smaller subunit> responsible for the accuracy of translation byensuring correct base-pairing between the codon inthe mRNA and the anticodon of the tRNA

3 binding sites for tRNA molecules:a.

A - binds an incoming aminoacyl-tRNA as directed bythe codon currently occupying the siteb. P - occupied by peptidyl-RNA, which carries the chain

of amino acids that has already been synthesizedc. E - occupied by the empty tRNA as it is about to exit

the ribosome

7. Transfer RNA

One specific type of tRNA is required per amino acidCarry a specific amino acid at its 3 endContains an anticodon

Anticodon> recognizes a specific codon on the mRNA> specifies the insertion into the growing polypeptide chainof the amino acid carried by that tRNA

> binding to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNAcodon is read 5-3 by an anticodon pairing in the flipped(3-5) orientation

Wobble hypothesis


26/29

> nontradional base-pairing between the 5 nucleotide (1 st

nucleotide) of the anticodon with the 3 nucleotide (lastnucleotide) of the mRNA codon> there need not be 61 tRNA species to read the 61 codonscoding for amino acids


27/29

Steps in Protein Translation:

1. InitiationAssembly of all the components of thetranslation system(ribosomes, mRNA, aminoacyl tRNA, GTP, initiator factors)

2. ElongationAddition of amino acids to carboxyl end of the growing chainRibosomes moves from the 5 end to the 3 end of the mRNARequires participation of GTP and elongation factorsFormation of peptide bonds is catalyze by peptidyltransferase activity of rRNA

3. TerminationRelease factor in eukaryote, eRF, recognizes all threetermination codons

Polysomes/ polyribosomeComplex of 1 mRNA and a number of ribosomes


28/29

Post-translational modification:1. Trimming2. Covalent modifications: phosphorylation, glycosylation,hydroxylation, carboxylation, farnesylation, acetylation

Consequences of altering the nucleotide sequence:1. Silent mutation

Codon containing the changed base may code for the sameamino acidEx: UCA UCU (still codes for serine)

2. Missense mutation

Codon containing the changed base may code for a differentamino acidEx: UCA (serine) CCA (proline)

3. Nonsense mutationCodon containing the changed base may become atermination codonEx: UCA (serine) UAA (stop codon)

4. Other mutations:a. Splice site mutations

Alter the way in which introns are removed from the pre-mRNA molecules thus producing aberrant proteinsEx: thalassemias

b. Frame- shift mutations

1 or 2 nucleotides are either deleted from or added to thecoding region of a message sequenceEx: Cystic fibrosis (due to a deletion of 3 nucleotides fromthe coding region of a gene, resulting in the loss of phenylalanine at the 508 th position)


29/29

c. trinucleotide repeat expansionA sequence of 3 bases that is repeated in tandem willbecome amplified in a number so that too many copies of thetriplet occur

Disease Repeatsequence

Normal # Diseasestate #

Location

1. Huntington CAG 11-34 42-100 Protein codingregion

2. Fragile X CGG 6-54 250-4000 5 untranslatedregion

3. Myotonic

dystrophy

CTG 5-30 >50 5 untranslated

region

Part 11 lecture (eukaryotic nucleus)

Documents

Transcript of Part 11 lecture (eukaryotic nucleus)