Exam 3 Human Genetics

Is DNA replication conservative or semiconservative ?

DNA replication is semiconservative, meaning each new double helix consists of one original and one newly synthesized strand.

What does it mean for dna to be semi conservative

It means that during DNA replication, each of the two resulting double helices contains one old strand and one new strand, preserving half of the original DNA.

in what direction does dna synthesis occur from the originof replication

bidirectional, synthesizing new DNA strands in a 5' to 3' direction.

what are some common features of eukaryotic origin of replication ?

• nearby AT rich seq

• GC strand slew

• G-quadruplexes

• Nucleosome free regions

what percentage of origins are used during any replication cycle?

only 10-20% of the origins are used

when does Pre-replication (preRC) assemble? what is it activated to form?

1. during G1 phase

2. activated to from pre-initiation complex (pre-IC) by recruitment of additional factors during S phase

   1. the active helicase that begins to unwind is formed from CDC45/MCMs/GINS

what are the components of the replication machinery?

• Dna helicase

• Dna topoisomerase

• ss binding protein

• dna primase

• dna polymerase

• dna ligase

what is the function of DNA helicase in replication ?

breaks the hydrogen bonds b/w dna strands to separate dna strands at origin

what is the function of dna topoisomerase in replication?

relieves surpercoiling (tangling) of dna ahead of the replication fork

what is the function of ss binding proteins in replication?

bind ss binding DNA to prevent strands from hydrogen bonding back together and to protect single strands

what is the function of dna primase in replication

synthesizes an RNA primer

short strands of RNA are 10-12 nucleotides long

what is the function of dna polymerase in replication?

forms phosphodiester bonds to link nucleotides

what is the function of dna ligase in replication?

seals okazaki fragments together

describe dna polymerase and dna synthesis?

• dna pol. can only add nucleotides to an existing strand

  • an RNA primer is need to primer replication

• dna pol. can only add nucleotides in the 5’-3’ direction on the new strand

• incoming dNTP binds to complementary base

• dna pol catalyzes formation of a phosphodiester bond b/w incoming dNTP and 3’-OH on previous dNTP

  • cleavage of a pyrophosphate provides energy for attachment.

describe the leading strand?

• dna primase lay down RNA primer

• dna pol. synthesizes a continuous DNA strand 5’-3’ moving in the same direction as the replication fork.

describe the lagging strand

DNA is synthesized discontinuously in short segments called Okazaki fragments, which are later joined together by DNA ligase. Each fragment requires an RNA primer for synthesis, with DNA polymerase ( synthesizes fragments ) adding nucleotides in the 5'-3' direction opposite of the replication fork.

what is each new dna strand formed from ?

combination of leading and lagging strands

describe what happens during the removal of primers

• rna primers must be removed

  • once an adjacent okazaki frag. has been synthesized, the primer is removed and replaced w/ dna nucleotides

  • once replaced by dna nucleotides, dna ligase forms the final bond to join okazaki frags.

what does dna ligase do to okazaki fragments?

joins them together

what are the 3 eukaryotic dna polymerase enzymes?

α - initiates dna replication in conjunction w/ primase

ε - replication of leading strand during S phase

δ - replication of lagging strand during S phase

describe dna pol. ε & δ

• processive enzymes

• don't dissociate after adding a nucleotide

• PCNA stabilizes dna pol on the dna template

what are rna primers removed by ?

flap endonuclease

• dna pol δ extends okazaki fragments

• runs into a primer & produces a flap

• flap endonuclease removes flap

• dna pol δ cont. to add nucleotides

• dna ligase links the fragments

Why is dna replication considered to very accurate ?

• dna pol. is unlikely to catalyze bond formation b/w adjacent nucleotides if a mismatched pair is formed

  • mismatches cause distortion of double helix

  • hydrogen bonding is less stable b/w mismatched pairs

• dna pol. has 3’-5’ proofreading mechanism ( can check for mistakes )

why can’t the 3’ ends of linear chromosomes be replicated? what resolves this issue?

• Because once the primer has been removed, there is no 3’ end for dna pol to add dna nucleotides to fill the gap.

• dna pol unable to replicate 3’ ends of template dna.

  • once terminal primer is removed, there’s no 3’-OH to add an incoming dna nucleotide to, results In chromosome shortening w/ each round of dna replication

• This issue is resolved by telomerase, an enzyme that adds repetitive nucleotide sequences to the 3' ends, preventing chromosome shortening.

Describe the function of telomeres

• consist of a short seq. repeated many times

• prevents loss of coding dna from the 3’ end of chromosomes during dna replication.

what does telomerase do ?

• has an rna template & reverse transcriptase activity to add dna nucleotides opposite the rna template nucleotides

• dna primase & dna pol. then fill the gap at the 5’ end of the new dna strand

what gradually happens to telomeres after birth?

• they gradually shorten with age in somatic ( body) cells that undergo cell division

  • in most somatic cells, telomerase stops being active after birth

  • remains active in cells w/ high rate of cell division, including activated B & T cells of the immune system, male germ line cells, and certain stem cells.

• somatic cells w/o active telomerase divide a finite number of times & then become senescent ( state where they cease to divide )

• most cancer cells have an active telomerase enzyme, which enables them to be immortal.

what is the central dogma?

• a theory that states that information flows from DNA > RNA > polypeptide

what is a gene ?

segment of dna that encodes a functional product

what is gene expression?

the info in a gene is expressed (I.e. used to make a functional product )

what percentage of genes encode polypeptides? what percentage of genes encode for functional RNAs?

• 90% polypeptdes

• 10% functional RNAs

what is a consensus sequence?

lists the most common nucleotide found at each position within the sequence

what 2 things do genes include?

1. seq. transcribed into mRNA

2. sequences that control expression of the gene

   1. Core promoters

   2. Regulatory elements

what are core promoters?

• site where RNA pol binds to initiate transcription

• about 1/3 of human promoters are focused promoters & about 2/3 are dispersed promoters

• bases are lableled in ref. to transcriptional start site

• up to 70% of promoters contain CpG islands

what regulatory elements?

distal (Far ) and proximal (Near) control elements

• bind transcription factors that control rate of transcription

describe transcription initiation

• RNA pol: enzyme that performs transcription

• General transcription factors ( GTFs):

  • TFIID binds to promote & recruits TFIIB

  • Rna pol II & TFIIF recruited next

  • Bind of TFIIE & TFIIH required for initiation

• mediator

  • large protein complex interacts w/ reg. TF’s.

  • involved in phosphorylation of CTD of rna pol.

describe transcription elongation?

• RNA pol. adds incoming RNA nucleotides to form an mRNA strand

  • synthesis in 5’-3’ directions

  • adds incoming NTPs to 3’ end

  • no primer required

  • U binds to A ( Instead of TA as in DNA)

  • lacks proofreading activity

• 3 diff pols.

  • RNA pol I: transcribes most rRNAs

  • RNA pol II: transcribes mRNA

  • RNA pol III: transcribes tRNA , SS rRNA

which strand is the mRNA seq. complementary to?

• template strand ( aka non coding strand )

which strand is mRNA seq the same as?

• exception U instead of T

• non template (aka coding strand)

in transcription elongation which direction is the mRNA always made to go?

5’-3’ direction

describe transcription termination

• terminates ~ 500 to 2000 bp downstream the polyA sequence ( serves as terminator seq when transcription ends )

  • proteins travel w/ RN pol direct mRNA cleavage

• Two models:

  • Allosteric: rna pol becomes destablized & dissociates

  • Torpedo: an exonuclease degrades RNA 5’-3’ after polyA signal

  • causes pol II to dissociate

what are the 3 mechanisms of pre-transcriptional gene regulation?

1. regulatory transcription factors

2. histone modificatons

3. dna methylation

what is a transcription factor?

Proteins that influence the ability of RNA polymerase to transcribe a gene

what are gen. transcription factors?

o Required for the binding of RNA polymerase to the promoter

o All genes transcribed by a particular polymerase require the same set of GTFs

o Bind to the promotor regio

what are regulatory transcription factors?

o Control the rate of transcription

o Different genes are controlled by different combinations of RTFs

o Approximately 2-3% of genes encode RTFs

o Bind to promoter proximal control elements and enhancers and silencers (distal control

elements)

in what 3 ways do regulatory transcription factors act to regulate the level of transcription from a gene?

o Enhance (activators) or inhibit (repressors) the binding of TFIID to the promoter.

o Interact with the Mediator complex to promote or inhibit its ability to phosphorylate RNA pol

o Interact with chromatin remodeling complexes to alter chromatin structure

what are enhancers and silencers bound by ?

regulatory transcription factors

Describe activators & enhancers

▪ Activators bind to enhancers to increase the rate of transcription (figure 1)

o Enhancers are located distally from the promoter (i.e., they are distal control elements). ▪ Activators may recruit TFIID

▪ Activators may interact with Mediator and cause Mediator to phosphorylate of the carboxyl tail

domain (CTD) of RNA polymerase II

describe repressors & silencers

▪ Repressors bind to silencers and decrease the rate of transcription

o Silencers are located distally from the promoter sequence (i.e., distal control elements). ▪ Repressors may inhibit the recruitment of TFIID

▪ Repressors may interact with Mediator to prevent phosphorylation of the CTD of RNA

polymerase II

Describe how transcription factors can recruit proteins that alter chromatin structure

remodels chromatin &uses ATP to do that

▪ ATP-dependent chromatin remodeling

complexes

o Change chromatin structure to make it more open or closed, which makes the DNA more accessible or less accessible, respectively, to transcription factors

What is the Histone code?

▪ Histone tails can be modified

o Pattern of modification is the ‘histone code’

▪ Acetylation

o Relaxes chromatin

o Associated with gene activation

▪ Methylation and phosphorylation

o Can be associated with activation or silencing depending on

the specific pattern of methylation/phosphorylatio

What does the methylation of dna of CpG islands typically cause?

gene silencing

• methylation of cytosine usually occurs in context of CpG sites

  • CpG islands occur in 70% of promoters

  • although most CpG islands aren’t methylated, methylation typically associated w/ gene silencing

    • often used to lock genes in off position

what are the 3 mRNA modifications?

▪ Addition of 5’ cap and 3’ polyA tail (all mRNAs)

▪ RNA splicing (most mRNAs)

▪ RNA editing (rare in human genes)

Describe 5’ capping in regards to RNA modifications

- A 5’ 7 -methylguanosine cap is added during pre-mRNA synthesis

▪ Bound by cap binding proteins for mRNA export from nucleus

▪ Recognized by translation initiation factors

- Polyadenylation adds a string of adenine (A) nucleotides after the polyA signal sequence

▪ Facilitates export from the nucleus

▪ Required for mRNA stability

Describe rna splicing in regards to RNA modifications

▪ Genes encoding polypeptides are composed of exons and introns

▪ Gene DNA sequence is transcribed into a pre-mRNA transcript containing introns and exons

▪ The spliceosome removes introns and splices together exons to produce mature mRNA

▪ Alternative splicing can produce different proteins from the same gene

what is the purpose of the spliceosome in rna splicing ?

❑ Spliceosome is composed of small nuclear RNAs (snRNAs) and proteins

❑ Conserved splice sites and branch sites are recognized by the spliceosome.

▪ Spliceosome removes introns and joins together exons

▪ Cuts the DNA at both splice sites and catalyzes a covalent bond between exons

▪ Alternative splicing is facilitated by splicing enhancers and repressors

what is RNA editing ?

• One type of base is converted to another

• Uncommon in human genes (fewer than 25 genes are edited in mammals)

what are the 2 mechanisms for post-transcriptional gene regulation?

▪ Control of mRNA stability

▪ Micro RNAs

Describe mRNA stability

mRNA stability is affected by the length of the polyA tail

▪ The rate of shortening of the polyA tail determines the lifetime of an mRNA

▪ mRNAs that are intended to be short lived have destabilizing elements, e.g., AU-rich

region (ARE)

▪ Proteins can bind to the ARE and either stabilize or destabilize the mRNA depending on

the conditions

Describe microRNAs

microRNAs (miRNAs) bind complementary mRNA and repress translation or promote degradation

❑miRNA usually has perfect complementarity to target mRNA at the seed region

▪ 1 miRNA can have many target mRNAs

▪ Estimated that 1/3 – 1/2 of the genome may be regulated by miRNAs

describe the flow of genetic material in gene expression

▪ DNA sequence is transcribed into an mRNA sequence

▪ mRNA sequence is read 5’ – 3’ as a series of triplet codons, starting at the start codon and

ending at the stop codon.

o Each codon binds to a complementary tRNA anticodon

o The tRNA carries the amino acid specified by the codon

the amino acid code is said to be _______. What does this mean?

degenerate

• same amino acid is specified by more than one codon

what does aminoacyl tRNA synthase do tRNA?

• adds amino acids to it

• Aminoacyl tRNA synthase adds amino acid to tRNA

  o A tRNA carrying its amino acid is referred to as a charged tRNA or an aminoacyl tRNA

what are the Wobble rules/

▪ Wobble rules allow the same tRNA to interact with more than 1 codon

o Therefore, we do not need 61 tRNAs.

o The table shows the original wobble rules proposed by Watson and Crick, which only

included standard bases, not chemically modified bases (e.g., 5-methyl uridine).

describe the open reading frame (ORF)

▪ Translation begins at the start codon (specifies Met)

▪ Translation ends at the stop codon.

▪ The open reading frame (ORF) of a gene starts at DNA encoding the start codon and ends at DNA encoding the stop codon.

▪ The coding sequence (CDS) includes only nucleotide sequences that are translated.

where does translation take place?

at the ribosomes

what 2 subunits do ribosomes have?

bacterial : large (50S )and small (30S)

eukaryotic: small (40S) and large (60S)

what are ribosomes made of?

rRNAs & proteins

describe the 3 ribosome sites ( APE site )

▪ A (aminoacyl) site

o Contains the incoming tRNA that has an anticodon complementary to the codon in the A site

▪ P (peptidyl) site

o Contains the tRNA holding the growing polypeptide chain

▪ E (exit) site

o The uncharged tRNA leaves the ribosome upon entering the E site

describe initiation of translation

▪ Initiation factor facilitates binding of small subunit to 5’ mRNA cap

▪ Initiation factor recruits tRNAMet to the P site

▪ 40S subunit scans the DNA for the first codon (Met)

o Okozak’s rules define the start codon consensus sequence

▪ 60S subunit binds to form the initiation complex.

▪ Initiation requires various initiation factors.

describe elongation in translation

▪ The same sequence of steps is repeated:

o Incoming tRNA binds to the A site.

o Peptide bond is formed to add the amino acid in the A

site to the end of the growing polypeptide chain in the P

site.

o The growing polypeptide is transferred to the tRNA in

the A site

o The ribosome moves one codon along to position the

next codon in the A site.

• The tRNA that was in the A site shifts to the P site

• The tRNA that was in the P site shifts to the E site and

describe termination of translation

The ribosome encounters a stop codon in the E site.

▪ The stop codon binds to a release factor

o Promotes release of the polypeptide and dissociation of the complex

what 3 things are req. for translation ?

Initiation, elongation and release factors

what determines the properties of an amino acid?

The side chain determines

what are amino acids linked by?

Amino acids are linked together by peptide bonds to

form polypeptides

what are the 2 terminuses of polypeptides?

Polypeptides have an amino terminus (N terminus) and a carboxyl terminus (C terminus)

▪ Amino acid sequence is always given N-terminus to C-terminus

o E.g., Met-Ser-Val-Tyr-Cys

primary structure of protein structure

Sequence of amino acids

secondary structure of protein structure

o Folding of local regions of the polypeptide chain

o Common structures are α helices and β sheets

tertiary structure of protein structure

o Overall 3D folded structure

o Final level of structure for proteins with one

polypeptide

quaternary structure of protein structure

o Association of two or more polypeptides

what is a mutation ?

permanent change in DNA sequence

what are point mutations ?

change to a single base pair

what are point mutations caused by?

base substitution:

• Transition mutation: purine → purine or pyrimidine → pyrimidine • Transversion mutation: purine → pyrimidine or pyrimidine → purine

what are silent mutations?

▪ A base substitution results in a synonymous codon, so there is no amino acid change

▪ Synonymous codons are different codons that encode the same amino acid

▪ Silent mutations do not typically affect phenotype

o Exceptions can occur if the silent mutation occurs in a splice site and affects splicing of the mRNA

what are missense mutations?

▪ A base substitution results in a non-synonymous codon and an amino acid change

▪ Missense mutations normally have a detrimental effect on phenotype because an amino acid change affects the folding and function of the resulting polypeptide.

o However, consequences can depend on the location of the mutation and the properties of the new amino acid.

▪ Occasionally, in the right environment, missense mutations can produce a protein with a new, beneficial function that is naturally selected.

what are nonsense mutations

▪ A base substitution results in a stop codon, which causes premature termination of translation and a shortened polypeptide.

▪ Nonsense mutations have a detrimental effect on protein function and therefore phenotype.

o The missing amino acids from the 3’ end will negatively affect the folding and function of the resulting polypeptide.

what are frameshift mutations? ( type of insertion deletion mutation )

▪ The reading frame of mRNA is defined by the start codon

o It is subsequently read as a series of triplet codons.

▪ The addition or deletion of nucleotides not divisible by 3 causes a frameshift mutation.

o It changes the codons, and therefore amino acid sequence, after the point of the addition or deletion (i.e., the reading frame is changed).

o Frameshift mutation has a detrimental effect on protein function and therefore on phenotype.

insertion/deletion mutations: nucleotides divisible by 3

▪ The insertion/deletion of nucleotides divisible by 3 results in the addition/deletion of amino acids (and possibly causes changes to the two flanking codons), but it does not cause a frameshift mutation.

▪ It usually has a neutral or detrimental effect on protein function (depending on the position, number and properties of the amino acids added/deleted).

describe mutations in noncoding regions

▪ Mutations in non-coding regions of a gene can also be detrimental by affecting gene expression or mRNA processing.

o E.g., mutations in the promoter or regulatory sequences may affect when and where the gene is expressed and/or the level of gene expression (i.e., how much gene product is produced.

o E.g., mutations in introns may affect splicing of the mRNA.

what is position effect?

gene expression is altered due to a change in the position of a gene on the chromosome.

o The DNA sequence of the chromosome has changed (i.e., it is a form of mutation), but the amino acid sequence of the gene product is unchanged.

▪ e.g., Relocation of the MYC gene from ch8 to ch14 changes the expression of the MYC gene in B-cells.

mutations may occur in what two types of cells?

1. germ line: cells that give rise to games ( egg & sperm cells )

2. somtic: body cells ( all cells that aren’t germ line cells)

what are spontaneous mutations?

mutations that result from natural processes.

what are 6 causes of spontaneous mutations?

1. errors in dna replication

2. crossing over b/w non homologous regions of chromosomes

3. spontaneous lesions

4. transposable elements

5. oxidative damage

6. replication of trinucleotide repeats

describe errors in dna replication

(not a common cause of mutation)

• DNA polymerases are very accurate and have proofreading activity

describe crossing over b/w non homologous regions of chromosomes

Results in deletions, duplications, inversions and translocations

describe spontaneous lesions

• Depurination: loss of a purine base from DNA

• Deamination: loss of an amine group from a base

• Tautomeric shift

describe transposable elements

mobile genetic elements that can move about the genome

what is depurination

loss of a purine base from DNA

▪ The chemical bond that links purine bases to the sugar group is prone to breaking.

o If the base is not restored prior to DNA replication, DNA polymerase could add any of the four bases opposite the empty site during DNA replication.

o 75% chance of introducing a base substitution

what is deamination & what is the result?

: loss of an amine group from a base ▪ The altered base does not follow the same base pairing

rules as the original base, resulting in a base

substitution during DNA replication.

o E.g., deamination of cytosine to uracil

o E.g., deamination of 5-methylcytosine to thymine

ex: causes a CG to TA transition ; CG changes to TG > TA

what is a tautomeric shift ?

o Bases exist in keto and enol or amino and imino forms

o Tautomeric shift is the change in structure of a base from common form to rare form

o Rare forms do not follow complementary base pairing rules

o A base substitution occurs if tautomeric shift occurs during DNA replication

Causes a TA to CG transition TA goes to TG → CG