genetics 3

What is Negative Supercoiling?

underwound DNA twist (opposite direction)

• helps create tension → makes DNA strands easier to separate

  • VITAL for Transcription & Replication

What is Positive Supercoiling?

overwound DNA twisting (same direction as helix)

• helps compact DNA & protect it from extreme physical conditions

  • excessive coiling → halts Transcription & Replication

What do Eukaryotic chromosomes consist of?

Single, linear double-stranded DNA molecules complexed w/ massive amounts of protein to form Chromatin

• protein → are Histones & Non-Histones

What are the Three Functional Sites of Eukaryotic Chromosomes?

• Centromeres

  • specialized DNA sequences → site for Kinetochore proteins

• Telomeres

  • specialized sequences located at ends of the chromosomes

    • protects from shortening, degradation, fusing with other chromosomes

• Origins of Replication

  • specific DNA sequences along chromosomes

    • DNA replication machinery binds/initiates

Which histones tend to form the Histone Octamer?

H2A, H2B, H3, and H4

How many histones are used to form the Histone Octamer?

2 copies of each core histone

What is H1 Histone?

the 5th Histone

• acts as a Linker Histone → is not part of the Octamer

• binds to the DNA entry/exit sites

• helps pack nucleosomes into tighter 30nm fibers!

Which type of DNA compaction state & transcriptional activity is known for being loosely condensed, transcriptionally active, and has unique or moderately repetitive across chromosome arms?

Euchromatin

What type of DNA compaction state & transcriptional activity is known for being highly condensed, transcriptionally permanently inactive, found consistently at centromeres?

Constitutive Heterochromatin

Which type of DNA compaction state & transcriptional activity is known for varying between states, can be active or inactive depending on cell type/development stage, and location varies by cell type?

Facultative Heterochromatin

What is Cohesin and what is its function?

multi-protein complex that contains SMC proteins

• found along entire length of each Chromatid until Prophase

• forms ring-like structure → holds Sister Chromatids together after replication

  • helps ensure proper alignment & tension on mitotic spindle

Promotes binding between Sister chromatids during Mitosis & Meiosis

What is Condensin & what’s its function?

large group of protein complexes → help compact & organize DNA when a cell divides

• binds to DNA & uses ATP hydrolysis to compact & fold Sister Chromatids tightly condensed

What are the 4 Types of Transposable Elements (TE)?

Insertion Sequences, Composite Transposons, Retrotransposons, and Simple Transposons

What is the Transposable Element: Insertion Sequence

short sequences that contain only genes necessary for transposition (Transposase gene)

• flanked by Inverted Terminal Repeats (ITRs) at the end

moves via conservative mechanism “cut and paste”

What is the Transposable Element: Composite Transposons

larger than IS → contain coding genes (Antibiotic resistance genes)

• flanked on both sides by Insertion Sequences (IS) → carry inverted repeats

moves via conservative mechanism “cut and paste” → mediated by Transposase

What is the Transposable Element: Retrotransposons

possess genes encoding Reverse Transcriptase & Integrase

• flanked by Long Terminal Repeats (LTRs) & non-Long Terminal Repeats (non-LTR)

moves via conservative “copy & paste” → replicate via RNA intermediate mechanism

• DNA element is transcribed into RNA intermediate

• reverse transcriptase converts RNA back into double stranded DNA

• integrase inserts new DNA into a new genomic location

What is the Transposable Element: Simple Transposons

flanked by Inverted Terminal Repeats (ITRs) & encode Transposases enzyme

move via conservative “cut & paste” → some can even replicate via cut & paste

What did the Meselson & Stahl experiment prove?

DNA replication is Semiconservative

• when DNA copies itself → the 2 original strands separate & each acts as a template to build a new complementary strand

results = each new DNA double helix contains one parental + one new synthesized strand !

How was the Conservative Model disproven by Meselson & Stahl?

it predicted that after 1 generation→ 2 distinct bands would be: 1 heavy and 1 light

• results showed → 1 single intermediate density band

How was the Dispersive Model disproven by Meselson & Stahl?

predicted that all subsequent generations would yield DNA molecules of mixed, uniform, hybrid density

• would gradually become lighter and lighter as generations passed

results showed after 2 generations → 2 bands: intermediate density & light density

DNA Helicase

binds to DNA, breaks hydrogen bonds between bases, & unwinds double helix ahead of replication fork

DNA Gyrase/Topoisomerase

alleviates positive supercoiling & torsional strain generated ahead of replication fork

• makes temporary cuts in DNA & reseals them

Primase

synthesizes short RNA primer complementary to single-stranded template

• provides free 3’-OH group for DNA polymerase → to build upon

DNA Polymerase III

primary replicative enzyme in bacteria

• synthesizes bulk of new DNA on both Leading & Lagging strands

  • adds nucleotides via 5’-3’ & reads template strand via 3’-5’

DNA Polymerase I

removes RNA primers on Lagging strand (using 5’-3’ exonuclease activity)

• replaces them with DNA nucleotides (5’-3’ polymerase activity)

DNA Ligase

catalyzes formation of a phosphodiester bond

• permanently seals & links adjacent DNA fragments together

  • DNA fragments are Okazaki Fragments

What is the difference between Leading & Lagging strands synthesis?

synthesized continuously in direction of replication fork while other is synthesized discontinuously in short pieces away from replication fork

• requires a single initial RNA primer at origin while other requires multiple RNA primers laid repeatedly

• DNa poly III extends strand smoothly; DNA poly I replaces primers w/ DNA & Ligase seals nicks

With DNA poly III, how is proofreading done?

polymerase pauses & 3’-5’ Exonuclease Domain → chews backwards & removes incorrect nucleotide

• continues in 5’-3’ synthesis

  • drastically lowers overall error rate!

Why is Telomerase needed for Eukaryotic Chromosomes?

important because rebuilds & maintains protective caps; it binds to G-rich 3’ overhang of telomeres → provides extra length

• allows primase & DNA poly III → fill in the Lagging strand & preserve chromosome length

What type of mutation substitutes a Purine for a Purine (A↔G) OR Pyrimidine for Pyrimidine (T↔C)?

Transition point mutation

What type of mutation substitutes a Purine for a Pyrimidine or vice versa (A↔C or G↔T)?

Transversion point mutation

What type of functional classification causes a nucleotide change that results in the same amino acid and no change in polypeptide?

Silent mutation

What type of functional classification causes a nucleotide change that results in a different amino acid being incorporated in the polypeptide?

Missense mutation

What type of functional classification causes a nucleotide change that converts an amino acid codon into a premature STOP codon?

Nonsense mutation

What type of functional classification causes a nucleotide change that inserts or deletes numbers of nucleotides not divisible by three, which completely shifts the reading frame & alters downstream amino acids?

Frameshift mutation

Which of the following mutations tend to affect negatively the protein functions?

Nonsense

Which mutation tends to stop translation?

Nonsense mutation → premature stop codon

Depurination

Spontaneous removal/hydrolysis of Purine base (A or G) from the DNA

• leaves a Apurinic (AP) site

Tautomeric Shift

Temporary change in base structure which alter base-pairing properties

• causes mispairing during Replication

  • stable form (Thymine & Guanine): Keto → can convert to Enol form

  • stable form (Adenine & Cytosine): Amino → but can convert to Imino

Ionizing Radiation (X-rays & Gamma rays)

High-energy radiation,that can penetrate deeply into biological molecules

• short wavelengths & high energy

• generates free radicals → cause double-stranded DNA breaks & base deletions

Non-Ionizing Radiation (UV light)

Lower energy, that CANNOT penetrate deep into biological molecules

• adjacent thymine bases are covalently linked → forming Thymine Dimers

Base Excision Repair (BER)

mechanism: corrects damage individual bases

• DNA N-glycosylase → recognizes abnomal base & cleaves bond between it

• DNA pol I & DNA Ligase → patches/fills gap & seals cuts

Nucleotide Excision Repair (NER)

mechanism: fixes bulky DNA distortions that alter double helix structure

• Enzyme complex → cuts short single-stranded patches containing lesion

• DNA pol & DNA Ligase → fills gaps & seals region

Mismatch Repair (MMR)

mechanism: scans newly synthesized using DNA polymerases for mismatched bases that escaped 3’-5’ proofreading

• recognizes unmethylated new strand → removes incorrect segment & resynthesizes it

Homologous Recombination Repair (HRR)

mechanism: repairs double-stranded breaks accurately using an identical Sister Chromatid as template

• highly error-free

Non-Homologous End Joining (NHEJ)

mechanism: repairs double-stranded breaks by directly sticking 2 broken ends back together w/o a template

• highly prone to errors!

  • may have→ small insertions or deletions

Promoter

is a specific DNA sequence/site that is located upstream of a gene

• RNA polymerase & transcription factors → tend to bind here to initiate Transcription

Terminator

a DNA sequence/site that signals RNA polymerase to STOP Transcription & release newly made RNA transcript

Activator

A regulatory protein that binds to DNA (Enhancer or Activator-binding site)

• to increase the rate of Transcription

Repressor

A regulatory protein, that binds to DNA (Operator or Silencer)

• to decrease or block Transcription

mRNA (Messenger RNA)

carries genetic code information thats transcribed from DNA out the nucleus to ribosomes

• for translation into proteins

rRNA (Ribosomal RNA)

structural & catalytic component of Ribosomes

• helps to form Peptide bonds between AA during Translation

tRNA (Transfer RNA)

adaptor molecule that binds a specific AA & delivers it to the Ribosome

• matching its anticodon to mRNA codon

Mechanism of Regulation of the Lac Operon with lactose

genes for lactose metabolism: lacZ, lacY, lacA → controlled by lac repressor & CAP

• NO lactose: lac repressor [ACTIVE] & binds to operator site

  • physically blocking RNA polymerase from Transcribing the operon [OFF]

• PRESENCE of lactose: Allolactose binds to repressor & releases the operator

  • allows RNA polymerase to bind & transcribe

Mechanism of Regulation of the Lac Operon with glucose

• NO glucose: intracellular cAMP rise & binds to CAP, both bind to promoter

  • ENHANCES RNA polymerase binding & increases transcription

    • Lactose + NO glucose = MAXIMUM EXPRESSION/TRANSCRIPTION

• with glucose: cAMP levels drop & CAP is inactive

  • transcription is low

Regulation of the Trp Operon (Repression & Attenuation)

Operon → responsible for synthesizing tryptophan (a repressible system)

• HIGH LVLS Tryptophan: acts as Corepressors & binds to inactive Trp repressor

  • activates repressor & binds to operator → STOPS TRANSCRIPTION

Attenuation regulation → operates via leader sequence containing tandem tryptophan

• abundant tryptophan: translation is fast, causes mRNA loop structure, acts as intrinsic terminator → halts Transcription

• scarce tryptophan: ribosome stalls at trp codons, forcing formation at different non-terminating loop, transcription continues

Cis-acting elements

DNA sequences located at SAME chromosome that regulates transcription

• ex. Promoters, Enhancers, Silencers

cannot diffuse & only influence genes physically linked to them

Trans-acting factors

Proteins or RNA molecules that are produced by genes elsewhere in genome & can diffuse through the cell → to bind to cis-acting elements

• ex. Transcription factors, Activators, Repressors

Alternative splicing

regulated process during gene expression where different combinations of exons joined together & introns are removed from a pre-mRNA

Within Alternative Splicing, what does the Repressors do?

“CUT THIS” → acts like roadblock that prevents that specific section onto the mRNA

• results: exon is skipped/left out of mRNA

Within Alternative Splicing, what does the Enhancer do?

“KEEP THIS” → acts like bright sign attracting machinery to take this to final mRNA

• results: exon is included in the mRNA

How does 5’ Cap & Poly-A tail helps to transport mRNA?

5’ Cap:

• added to the end during transcription

• protects mRNA from rapid degradation

• assists in export out of nucleus

• required for binding of mRNA to Ribosome during Translation

Poly A tail

• enhances mRNA stability in cytoplasm

• aids in export from nucleus

• facilitates translation efficiency

What happens if there is a STOP codon is located at the starting site?

rather than being AUG → STOP codon at the beginning causes Translation to not begin

Given 70% G and 30% U are mixed together, what percentage of the radiolabeled AA will be glycine (GGU or GGG)

70% → 0.7 GGG: (0.7)(0.7)(0.7)= 0.34 → 34%

30% → 0.3 GGU: (0.7)(0.7)(0.3)= 0.15 → 15%

---

34 + 15 = 49%

Wobble Hypothesis

explains how certain tRNA can pair with more than one codon due to flexible base pairing at the third position of the codon, allowing for a more versatile and efficient translation process.

• 3rd nucleotide of mRNA codon & 1st nucleotide of tRNA anticodon is flexible (wobbly)

• 1st 2 bases of the codon→ strict/precises pairing w/ bases of anticodon of tRNA

  • allows a single tRNA to recognize more than 1 codon

What is the difference in Translation Initiation of Bacteria vs. Eukaryotes?

Bacteria

• tends to initiate by ribosomes binding in the Shine-Dalgarno sequence before start codon

• 1st AA → Formyl-methionine (fMet)

• happens in cytoplasm & same time as Transcription

Eukaryotes

• ribosomes bind at the 5’ Cap & Kozak consensus sequence

  • subunits bind to Cap & scans downstream until 1st AUG start codon

• 1st AA→ Methionine (Met)

• transcription happens in nucleus & translation in cytoplasm

During the Elongation stage of Translation, which ribosomal site receives the incoming charged tRNA and matches its anticodon to the mRNA codon?

A site (Aminoacyl site)

During the Elongation stage of Translation, which ribosomal site holds the tRNA carrying the growing polypeptide chain?

P site (Peptidyl)

During the Elongation stage of Translation, which ribosomal site hold the uncharged tRNA right before it is released from the ribosome?

E site (Exit)

What is the Termination stage of Translartion?

when a ribosome encounters a STOP codon → UAA, UAG, UGA

• within the A site

• no matching AA tRNA exists

• Release factors bind to A site → hydrolyzes bond between polypeptide chain & final tRNA

  • releases completed protein & causes ribosomal subunits to dissociate