CELS FINAL EXAM

Structure of DNA

helical structure
double-stranded helix
bases perpendicular to the length of the DNA molecule
diameter of the helix is constant

provides stimulus for deciphering genetic code

Relationship between purines and pyrimidines

Pyrimidines are 1 ring structrues C and T- they connect to 2 ring structures A and G

Formation of a polynucleotide

Nucleotide monomers are joined together to create a phosphodiester bond to form a nucleic acid

Phosphodiester Bond

OH group of the 3rd carbon of one nucleotide reacts with the phosphate group attached to the 5th carbon on another nucleotide

Antiparalel Strands

5’-3’ and

3’-5’

antiparallel strands form the double stranded helix

The nucleobases point towards the middle

always synthesised in the 5’-3’ direction

Replication of DNA

semi conservative

each dna strand of the double helix is used as a template strand for the synthesis of two new strands

each daughter strand of DNA conserves half of the original DNA

The Central Dogma of Molecular Biology

DNA is transcribed into mRNA and translated to a protein

Gene expression

The process by which information from a gene is used in the synthesis of a functional gene product: a protein or non coding RNA

Gene

a defined region of DNA that produces a type of RNA molecule that has some function

DNA may contain sequences that:

are responsible for the regulation of the synthesis of RNA

produce RNA

are responsible for the further processing of the RNA

Transcription

The synthesis of RNA from DNA catalysed by RNA pol

RNA Polymerase in transcription

catalyses the extension of the 3’ end of an RNA strand by one nucleotide at a time

forms an RNA molecule by catalysing the formation of phosphodiester bonds between ribonucletides

selects the correct nucleotides to incorporate into RNA based on the sequence being transcribed

RNA Pol I

catalyses transcription

has primase function so initiatsed a chain de novo

Coding and Template Strands

Coding strand is in the 5’-3’ direction and the template strand is in the 3’-5’ direction

The template strand is read and mRNA is transcribed from it resulting in a newly synthesised strand that is identical to the coding strand

Stages of transcription

initiation

elongation

termination

Initiation of transcription

transcription factors bind to the TATA box and other regions of the promoter

RNA pol II binds to transcription factors forming a transcriptional initiation complex with the transcription factors

RNA pol II recruits helicase

two DNA strands seperate and RNA pol II starts mRNA synethsis

Helicase

binds to the AT rich region of the promoter to start unzipping DNA

Elongation of transcription

RNA pol II uses the template strand which runs the the 3’-5’ direction as a template inserts complementary RNA nucleotides in the 5’-3’ direction

Topoisomerase II acts

Parental DNA strands bind back together

Topoisomerase II

an enzyme that releases tension that builds up ahead of RNA pol II

Anatomy of Eukaryotic genes

The promoter region contains the TATA box/ the AT rich region

The coding sequence is non continous and is surrounded by the 5’ UTR (untranslated region) and the 3’ UTR

The 5’ UTR is connected to the 5’ G cap

The 3’ UTR is connected to the Poly-A tail

Splicing

The coding sequence is made of introns and exons

The introns are intervening sequences which are removed through splicing

Exons join together to give proteins

Coding sequence

portion of a gene’s DNA translated into a protein

Promoter

DNA segment recognised by RNA polymerase to initiate transcription

UTR

transcribed but not usually translated

contain regulatory elements that influence gene expression at transcription

5’ UTR

facilitates the addition of the 5’ G cap

3’ UTR

facilitates the addition of the poly-A-tail

5’ G cap

prevents mRNA degradation, promotes intron excision and provides a binding site for the small ribosomal subunit

Poly-A tail

prevents mRNA degradation and facilitates export of the mRNA from the nucleus to the cytoplasm

Differences between prokaryotic and eukaryotic transcription

In prokaryotes: transcription occurs in the cytoplasm

translation and transcription and coupled

In eukaryotes: transcription occurs in the nucleus and translation occurs in the cytoplasm

Transcription and translation are not coupled

Codon

triplet of bases which encodes for one amino acid

features of the genetic code

61 out of 64 amino acids code for an amino acid

3 codons code for the stop of protein synthesis

UAA, UAG, UGA

codon coding for the start of protein synthesis

AUG- codes for the amino acid methionine

signals the start of translation and tells the ribosome where to begin assembling the amino acid chain

AUG is the first codon read when synthesising a protein

Methionine

every newly synthesised protein starts with methionine

when ribosomes read AUG, it brings a tRNA carrying methionine

tRNA

small single strand RNA adaptor molecule

each tRNA has a region which can bind an amino acid and a region which can interact with mRNA

3 key features of the tRNA

amino acid attachment site- interacts with mRNA to obtain information

Anticodon- 3 nucleotides which interact with codons

3D structure- makes adaptor molecule

Charging tRNA

an enzyme recognises both a specific amino acid and the correct tRNA for this amio acid and joins them together- there are 20 different enzymes one for each amino acid

Translation

the synthesis of proteins by ribosomes using mRNA as a set of instructions

Locations of ribosomes

Bound to the rER- synthesise proteins that are used within the plasmamembrane or exocytosed from the cell

Free in the cytosol- synthesise proteins that are released into the cytoplasm within the cell

charged tRNA

tRNA with a specific amino acid already attached ready to be delivered to ribosomes for protein synthesis

3 stages of translation which require energy input

initiation elongation and termination

initiation of translation

a specfic initiatino tRNA carrying methionine binds to the ribosomal subunit

The small ribosomal subunit and initiator tRNA complex identifies the 5’ G cap and attaches to the mRNA

The small ribosomal subunit and initiator tRNA complex moves along the mRNA in the 5’-3’ direction until it finds the 5’-3’ AUG start codon- the complimentary 3’-5’ UAC anticodon of the tRNA binds to the start codon

the initiator tRNA carrying the first methionine is positioned in the P site

the large ribosomal subunit attaches and the initiation complex stops

Elongation

a charged tRNA with an anticodon complimentary to the A site lands in the A site

The ribosome will break the bond that binds the amino acid to the tRNA in the P site and transfer the amino acid to the newly arrived amino acid attached to the tRNA in the A site, this forms a peptide bond between the tRNA now with the growing amino acid chain in the A site and the uncharged tRNA in the p site

While the tRNAs are bound to the mRNA in the P and A sites the ribosome moves three nucleotides down the mRNA

the tRNA with the growing amino acid chain moves from the A site to the P site so the chain of amino acids can exit through the tunnel located above the P site and the uncharged tRNA moves from the p site to the E site

In the E site the anticodon of the tRNA detaches from the mRNa codon and the uncharged tRNA is expelled

A new charged tRNA with an anticodon complementary to the next A site codon enters the ribosome at the A site and the elongation process repeats itself

Termination of translation

When the ribosome reaches a stop codon and protein called the release factor enters the A site

The release factor breaks the bond between the P site tRNA and the final amino acid, using water (promoting hydrolysis)

This causes the polypeptide chain to detach from its tRNA and the newly made polypeptide is released

The small and large ribosomal subunits dissociate from the mRNA and eachother

Direction of DNA or RNA synthesis

5’-3’ direction

The parental Template strand runs in the 3’-5’ direction

Eukaryotic DNA replication

multiple large linear chromosomes

multiple origins of replication as the whole chromosome is too large to be replicated as a whole

replication is bidirectional

semi discontinous replication

leading strand is continously synthesised in its 5’-3’ direction

lagging strand is discontinously synthesised in its 5’-3’ direction as okazaki fragments

The direction of DNA synthesis is in towards the replication fork

Origin of Replication

AT rich regions where DNA strands are easier to pull apart due to less H bonds

Primase

a type of RNA polymerase enzyme that makes an RNA primer acting as a starting point for DNA polymerisation

a starting point for nucleotide addition catalysing the formation of a phosphodiester bond to form a nucleotide

DNA Pol III

Enzyme that synthesises a new DNA strand by adding nucleotides complementary to the parental template strands- progressive addition of new nucleotides

needs and OH group onto which the phosphate group of the incoming nucleotide can be attached

only makes DNA in the 5’-3’ direction

cannot bind to single stranded DNA and start copying it

DNA Polymerase I (2 activities)

RNase- RNase H is an endonuclease enzyme that recognises DNA:RNA hybrids and degrades the RNA part

DNA Polymerase: synthesis DNA by adding nucleotides complementary to the parental DNA template of the lagging strand

DNA Ligase

joins newly synthesised okazaki fragments together creating phosphodiester bonds once the RNA primers and been removed and replaced ny nucleotides

also joins newly synthesised fragments from multiple replicatin bubbles including the leading strands

Polymerase Chain Reaction PCR

In labratory test tube (vitro) DNA replication

millions to billions of copies of a particular DNA section from a very small original amount can be studied in greater detail

when are DNA errors repaired?

during replicatin- exonuclease

after replication- endonuclease

Accuracy of DNA replication

DNA pol III is highly accurate

DNA pol III has a proofreading mechanism checking newly inserted nucleotide bases against the template strand

error occurs 1 in 10^8-10

Exonuclease activity of DNA pol III

incorrect bases are removed by a 3’-5' activity of the exonuclease

occurs at the outer ends of dna strands

Repair of errors after DNA replication

errors can occur through incorrectly inserted bases not corrected by DNA pol III- radiation damage or natural and chemical modification of bases

removed by an endonuclease (occurs within the DNA strand)

A DNA polymerase makes new DNA

DNA ligase joins new DNA to existing DNA

importance of correcting errors

if not corrected it becomes a part of the DNA template leading to a permanent change in DNA and thus a mutation

how do eukaryotic cells store genetic information

linear chromosomes

double stranded DNA genome type

how do prokaryotic cells store genetic information

circular chromosomes

double stranded DNA genome type

how do acellular microbes store genetic information

chromosomes are linear, circular, or segmented

genome type is double stranded DNA or RNA

or single stranded DNA or RNA

Karyotype

ordered visual representation of chromosomes in a cell- ordered by size- all chromosomes are present in homologous pairs

image is used from metaphase of cell division when the chromosomes condense so they can be seen

Cell cycle

the growth period Interphase is the longest stage made up of three stages and alternates with the mitotic phase

Interphase

G1 phase- metabolic activity and growth

S phase- metabolic activity, growth and DNA synthesis

G2 phase- metabolic activity, growth and preparation for cell division

Duplicated Chromosome

comprises of 2 genetically identical sister chromatids which seperate during mitosis

each chromatid is made of a double stranded DNA molecule

sister chromatids are joined together at the centromere

G2 of interphase

nuclear envelope still intact

nucleolus is visible

two centrosomes form

duplicated chromosomes cannot be been individually as they have not yet condesed

Mitosis

the production of two genetically identical daughter cells

Prophase

nucleoli disappear

Duplicated chromosomes condense and appear as two identical sister chromatids joined at their centromeres

Mitotic spindle begins to form

Microtubules lengthen, and the centrosomes move to opposite poles

Prometaphase

nuclear envelope breaks down and the chromosomes fully condense

a protein structure known as a kinetochore forms at the centromere of each chromatid

microtubules attach to the kinetochore forming kinetochore microtubules

nonkinetochroe microtubules lengthen the cell by interacting with those from the opposite pole of the spindle

Metaphase

centrosomes are now at the opposite poles of the cell

kinetochore microtubules are attached to the kinetochores of all sister chromatids

duplicated chromosomes align at the metaphase plate and homologous pairs do not interact

centromeres lie on the plate an equal distance between the spindle’s two poles

Anaphase

sister chromatids disjoin at the centromeres

sister chromatids separate during anaphase and each chromatid becomes a daughter chromosome

daughter chromosomes move towards opposite poles as their kinetochore microtubules shorten

the non kinetochore microtubules lengthen and the cell elongates

anaphase ends when the two poles of the cell contain identical and complete collections of chromosomes

Telophase

chromosomes become less condensed

spindle microtubules break down

two daughter nuclei with nuclear envelopes form in the cell

nucleoli reappear

two genetically identical nuclei are produced

Cytokinesis

the cytoplasm divides into two daughter cells

in animal cells a cleavage furrow pinches the cell in two

in plant cells a cell plate is formed between the daughter cells

each daughter cell has one copy of each duplicated chromosome

sexual cycle

found in most eukaryotes

offspring is not genetically identical to parent

what happens if gametes were produced by mitosis

gametes would be diploids and resulting embryos would be tetraploid (4n)

Meiosis

cell division in sexually reproducing organisms

consists of two rounds of cell devision but only one round of dna replication

halves the number of chromosomes going into gametes so the diploid number is retained in zygotes

results in 4 genetically distinct haploid cells (gametes)

Meiosis I

homologous pairs of chromosomes are seperated

Prophase I

nuclear envelope breaks down

chromosomes condense

spindles form

crossing over between non-sister chromatids occurs at chiasmata

each chromatid is now a mix of DNA from each homologous chromosome

Metaphase I

chromosomes are attached to the kinetochore microtubules at each centromere

each pair is lined up independently

paired homologous chromosomes have moved to the metaphase plate

chiasmata line up on the metaphase plate not centromeres like mitosis

Anaphase I

recombined homologous chromosomes disjoin

sister chromatids remain attached to each other

nonkinetochore microtubules extend so the cell alongates and each duplicated chromosome moves to the poles at opposite ends of the cell

Telophase I and cytokinesis

duplicated chromosomes reach the poles at opposite ends of the cell

spindle disappears and nuclear envelope reforms

cytoplasm divides, resulting in two haploid cells from the diploid parent cell, each new cell contains half of the genetic information these cells are genetically different due to crossing over

Meiosis II

sister chromatids are seperated

Prophase II

spindle forms as the centrosomes duplicate and move to opposite poles

kinetochroe microtubules attach to each duplicated chromosome at the centromere via the kinetochroe proteins

each duplicated chromosome is still composed of two chromatids attached at centromeres

metaphase II

duplicated chromosomes align at the metaphase plate

centromeres lie on the metaphase plate

anaphase II

sister chromatids disjoin at the centromeres

each chromatid becomes and independant daughter chromosome

daughter chromosomes move towards opposite poles as their kinetochore microtubules shorten

the nonkinetochore microtubules lengthen and the cell elongates

telophase II and cytokinesis

two daughter nuclei with nuclear envelope form in the cell

each of the two daughter cells produced has a haploid set of unduplicated chromosomes

sexual reproduction produces genetic diversity through:

independent assortment of chromosomes

crossing over

random fertilisation of gametes

genetic diversity allows selective responses to:

spatially variable environments eg climate

changing environments eg seasons

sib-sib competition

no. chromosome pairs in relation to no. gametes

the number of possible gametes is 2^ number of chromosome pairs

meiotic nondisjunction

failure of chromosomes to disjoin during meiosis

in meiosis I homologous chromosomes do not seperate

in meiosis II sister chromatids do not seperate

aneuploidy

meiotic nondisjunction results in some gametes recieving an adnormal number of a particular chromosome

when these gametes use with a normal gamete the resulting zygote will also have an abnormal number of a particular chromosome

monosomy

Fertilisation involves a gamete with no copy of a particular chromosome, and a regular gamete will result in a monosomic zygote with only one copy of a chromosome (2n-1)

trisomy

zygote contains three copies of one chromosome (2n+1)

survival of aneuploid organisms

the gametes will not implant properly and the cells will not properly divide

the zygotes will often spontaneously abord

if they survive there will be observable defects

down syndrome

trisomic autosomal aneuplod condition

3 copies of chromosome 21

karyotypes have 47 chromosomes

klinefelter syndrome

XXY

trisomic aneuploid condition of a sex chromosome

individuals have an extra copy of X chromosome

karyotypes have 47 chromosomes

turner syndrome

monosomic aneuploid condition of a sex chromosome

individuals have only one chromosome X

karyotypes have 45 chromosomes

polyploidy

possession of more than two complete sets of chromosomes

most are phenotypically normal as one extra chromosome seems to disrupt the genetic balance more than a complete set

how does polyploidy arise

may arise due to nondisjunction of all chromosomes in one gamete or the failure of a diploid zygote to divide after replicating its chromosome in the G2 phase