molecular genetics exam 3 study guide

what are the causes of DNA damage?

1. -Chemical/physical-oxidative reactions/toxic oxygen species(ROS).

-free radicals(byproducts of metabolism) attack DNA to scavenge electrons and cause breaks in the phosphate backbone , ionizing radiation produces free radicals.

-environmental chemicals(exogenous)

2. antioxidants have extra electrons that they can donate to neutralize free radicals

deamination definition and example

hydrolytic removal of an amino group(-NH2). ex. cytosine—>uracil created U-G mismatch (U is preferentially removed), but if its not then C—>T transition after replication can cause mutation

what happens from pyrimidine dimer formation (UV damage)?

UV light causes covalent bonds between adjacent pyrimidines usually T-T dimers which distorts DNA helix shape and replication stalls. only fixed by NER

What is the ‘backup’ for NER failure?

specialized polymerase that can replicate past T-T dimers and insert A-A opposite the dimer-BUT for CC or TC dimers the 3’cytosine can deaminate to uracil leading to C—>T transition

What is depurination and its consequences?

loss of a purine base(A or G) due to cleavage of glycosidic bond at a rate of ~5000 per cell per day. this can potentially stall replication or skip the missing base causing single base-pair deletion only repaired by BER

Causes of Double stranded breaks?

Replication errors, ROS, ionizing radiation, UV, oxidation

the most common UV lesion is___ which is repaired by____

thymine dimer, NER

depurination removes___

A or G

Deamination of cytosine produces___

Uracil

C—>T mutation type is___

transition

NER disease ___

XERODERMA PIGMENTOSUM

Backup polymerase for T-T dimers is DNA____

polymerase η

___is the most frequent spontaneous DNA damage

Depurination -5000/day

T/F:deamination converts cytosine to thymine

FALSE CONVERTS CYTOSINE—>URACIL

T/F:NHEJ requires a homologous chromosome

FALSE

T/F:UV light causes pyrimidine dimers

true

T/F:Polymerase η can bypass T–T dimers

TRUE

T/F: XP patients lack functional NER

TRUE

what are transposable elements?

mobile genetic elements that can move to new genomic locations.

______are a major source of mutation, genome rearrangement, and genome expansion

transposable elements

Nearly 50% of the human genome is derived from transposable elements: 20.4% from___, 13.1% from___, 8.3%from ___, and 2.8% from___

LINE,SINE,LTR retrotransposons, DNA transposons

Subtypes of Class I retrotransposons (copy and paste)

LTR retrotransposons, non-LTR transposons (LINE’s), SINE’s

T/F Class II DNA transposons are only found in eukaryotes

FALSE found in both

Class II DNA transposons (cut and paste and copy and paste) move as DNA, not RNA , and require___

transposase

Autonomous encode their own___

transposase

Class __ elements move via an RNA intermediate

I

Class __ elements move as DNA

II

___binds Terminal inverted repeats (TIRs)

Transposase

____form during insertion

Target site duplications(TSD)

non-replicative transposition= __and__

cut and paste

Replicative transposition= __and_

copy and paste

____ use virus like particles(VLP’s)

LTR retrotransposons

LINE-1 encodes ___ and ___

ORF1 and ORF2

SINEs are non-autonomous and require ___machinery

LINE

__ is the most abundant SINE in humans

Alu

T/F LTR retrotransposons use reverse transcriptase.

TRUE

T/F LINE’s are autonomous retrotransposons

true

T/F replicative transposition leaves the original copy in place

true

the enzyme in transcription in bacteria is__

DNA-dependent RNA polymerase

TATAAT consensus region=

-10 region

TTGACA consensus region=

-35 region

bacteria promotor functions:

positions RNA polymerase, determines transcription frequency, recognized by sigma factor

abortive initiation:

RNA polymerase repeatedly synthesizes short 2–9 nt transcripts that are released.

• Occurs while polymerase is still bound to the promoter.

• Happens before polymerase escapes the promoter.

Promoter clearance:

RNA polymerase breaks interactions with promoter DNA and sigma factor.

• Transition from initiation → elongation.

• Sigma factor is released.

Sigma factors determine which promoters___ recognized

RNA polymerase

Holoenzyme structure:

Core+sigma factor

in bacteria, ____ and ___ occur simultaneously

transcription and translation

monocistronic:

encodes one protein

T/F sigma factor increases promotor specificity

TRUE

Rho binds at the ___site

rut

T/F Rho‑independent termination requires a hairpin.

TRUE

Difference between prokaryotic and eukaryotic transcription

Eukaryotic DNA is packaged into chromatin and require basal transcription factors to bind first

assembly of the pre-initiation complex(PIC)

Core promotor usually contains TATAAA box , RNA Pol I binds only after basal factors assemble

promotor clearance requires:

TFIIH helicase activity

• CTD phosphorylation

why does pausing occur?

• Allows fine‑tuning of transcription.

• Acts as a checkpoint for proper 5' capping.

• Important in developmental gene regulation.

distal control elements types:

• Enhancers → increase transcription.

• Silencers → repress transcription.

• Can be upstream, downstream, or within introns.

• Can function far from the promoter.

Proteins that bind them

• Activators (bind enhancers).

• Repressors (bind silencers).

• The first basal factor to bind the promoter is

TFIID

Enhancers bind ___;silencers bind___

activators,repressors

T/F TFIIH unwinds DNA during initiation

true

DNA methylation

• Methylated CpG islands → transcription OFF 

• Unmethylated CpG islands → transcription ON

Eukaryotic pre‑mRNA undergoes three major processing steps before it becomes a mature mRNA: 

1. 5′ capping 

2. 3′ cleavage and polyadenylation (poly‑A tail) 

3. RNA splicing (including alternative splicing)

ALL OCCURS IN NUCLEUS

When do mRNA modifications take place?

5’capping-partway through transcription, polyadenylation-at the 3’end of the pre-mRNA after RNA Pol II encounters the poly(A) signal,splicing-occurs before the active spliceosome forms and at the 5’splice site GU

Distinctive features of the cap:

• 5'–5' triphosphate linkage (unique to eukaryotes).

• 7‑methylguanosine.

• Required for:

• mRNA stability

• splicing of the first intron

• nuclear export

• translation initiation (via eIF4F complex)

steps of 5’cap formation

1. The first nucleotide of mRNA is almost always a purine (A or G).

2. A guanine nucleotide is added to the 5' end via a 5'–5' triphosphate linkage.

   • Enzyme: Guanylyltransferase (GT)

3. The guanine is methylated at N‑7 → 7‑methylguanosine (m⁷G).

4. Additional methylations may occur on the first or second nucleotide (Cap 1, Cap 2)

Proteins that bind the cap

• In nucleus: CBP20/80 (cap‑binding complex)

• In cytoplasm: eIF4F (eIF4E + eIF4G + eIF4A)

3’polyadenylation poly A tail formation required RNA sequence elements:

1. AAUAAA — Polyadenylation signal (PAS)

2. Cleavage site — 10–30 nt downstream of AAUAAA

3. GU‑rich DSE (downstream sequence element)

4. USE (upstream sequence element)

steps of poly-A tail formation

1. RNA Pol II transcribes AAUAAA.

2. A protein complex binds:

   • CPSF (cleavage and polyadenylation specificity factor)

   • CstF (cleavage stimulation factor)

   • Endonuclease (CPSF‑73)

3. RNA is cleaved between AAUAAA and the GU‑rich DSE.

4. Poly(A) polymerase (PAP) adds ~50–250 adenines without a template.

5. PABP (poly‑A binding protein) binds the tail and stabilizes mRNA.

functions of the Poly-A tail

• Protects mRNA from degradation.

• Required for translation initiation (PABP interacts with eIF4G).

• Removal of the tail → rapid degradation.

Splicing required sequence elements:

Splicing removes introns and joins exons.

Required elements

1. 5' splice site — GU

2. Branch point A

3. Polypyrimidine tract

4. 3' splice site — AG

formation of the commitment complex(early spliceosome assembly)

This occurs before the active spliceosome forms.

Steps

1. U1 snRNP binds the 5' splice site (GU).

2. SF1 (branch point binding protein) binds the branch point A.

3. U2AF binds:

• U2AF65 → polypyrimidine tract

• U2AF35 → 3' splice site (AG)

This forms the commitment complex (also called E complex).
It “commits” the pre‑mRNA to the splicing pathway.

spliceosome assembly and lariat formation steps

1. U2 snRNP replaces SF1 at the branch point A.

2. U4/U5/U6 tri‑snRNP joins.

3. U6 replaces U1 at the 5' splice site.

4. Catalysis begins:

• Branch point A attacks the 5' splice site, forming a lariat (2'–5' phosphodiester bond).

• 3' OH of exon 1 attacks the 3' splice site, joining exons.

Result

• Introns removed as a lariat.

• Exons ligated to form mature mRNA.

alternative splicing and drosophila sex determination why it matters:

• Shows how alternative splicing can produce different proteins from the same gene.

• Demonstrates how splicing controls developmental fate.

T/F AAUAAA is required for cleavage and polyadenylation.

TRUE

T/F Alternative splicing can produce different proteins from the same gene. —

TRUE