BMED 3600 Exam 2 Review: DNA Replication, Transcription & RNA Processing, Gene Regulation...

1. The parent strand splits

2. New strand is made complimentary to the direction of the initial strand (5' to 3')

Explain how copies of DNA are made?

5' to 3' directionality

____________________ determines synthesis of DNA

size

Incorrect base pairs slow strand progression via polymerase due to _____ difference.

Arg and Gln, the molecular ruler

Base pair length is measured by ______________ in polymerase

There's so many checks and balances in the body during DNA synthesis constantly that mistakes are unlikely

Why our cells do not mutate rapidly

5' to 3' polymerization, 3' to 5' exonucleolytic proofreading, strand-directed mismatch repair

The 3 steps that give rise to high-fidelity DNA synthesis

1. Initiator proteins bind to and destabilize the AT rich sequence in the replication origin

2. DNA helicase binds to the replication origin(s) and opens the helix by spinning so it becomes linear

3. Helicase is activated

4. DNA primase binds at the replication fork

5. RNA primer bind at the replication fork(s)

6. DNA polymerase binds and begins synthesis of new DNA chains:

The leading strand synthesizes continuously in the 5' to 3' direction following the helicase and the lagging strand synthesizes discontinuously (Okazaki fragments) in the 5' to 3' direction going against the direction of the helicase

7. DNA polymerase and ligase join the Okazaki fragments together to create one continuous strand

8. Everything detaches once replication is completed

The process of DNA replication

monomers come together with a catalyst to start a reaction, the reaction continues until stopped

How polymerization reactions start

backstitching

The lagging strand undergoes a ______ mechanism

1. Denaturation: increase temperature, strands separate

2. Annealing process: reduce temperature, add primers, let primers bind

3. Extension: add DNA polymerase, DNA synthesis w/o lagging strands

The basic PCR method

amplifies specific cDNA from mRNA

Reverse transcriptase PCR (RT-PCR) _____________ in 4 steps

further cloning/analysis, diagnose infections, quantify mRNA

RT-PCR is used to obtain cDNA for ___________

RNA

RT-PCR is commonly used in ___________ analysis and pathogen testing (COVID)

1. Extract mRNA

2. RT reaction

3. remove mRNA

4. PCR of the target gene

The steps of RT-PCR

mRNA level quantification and COVID

quantitative reverse transcriptase PCR (qRT-PCR) is used for accurate _______ and _______ testing

in vitro

PCR is a DNA replication _____ through steps of denaturation, annealing, and extension

further use/detection

RT-PCR makes a cDNA copy from mRNA and amplifies it for ______

nuclear periphery

segregation of heterochromatin towards the _________ is found in nearly all animal cells

tri-methylation through microscopy

chromatin is imaged with ________

spacial organization

_______ has consequences for cell differentiation

DNA > RNA > protein

the central dogma of molecular biology

miRNA

regulate gene expression by blocking translation of specific mRNAs and cause their degradation

siRNA

turn off gene expression by directing degradation of selective mRNA's and establishing compact chromatin structures

gene silencing

Small interfering RNAs (siRNAs) bind to mRNA, which is then destroyed by RNA-induced silencing complex (RISC)

ribonucleoside triphosphate uptake channel

forms RNA units

1. promoter binding

2. initiation

3. elongation

4. termination

the 4 steps of transcription for prokaryotes and eukaryotes

transcription factor

a _____ is a factor that helps with the transcription process

GC rich region

primase binds here

The orientation of the promoter sequence

How do we now which is the coding and which is the template strand?

1. RNA polymerase binds loosely to DNA and slides along it, the promoter region is recognized by the sigma factor and binds tightly to the DNA,

2. At the promoter level, there is alteration between closed and open complex

3. Abortive initiation begins the process of RNA production and incorrect RNA is ejected and degraded

4. Productive initiation begins and the sigma factor is released to initiate elongation

5. The transcript is elongated

6. A hairpin structure forms to terminate the transcription process

7. DNA helix, single stranded RNA, and RNA polymerase are released

8. The sigma factor and RNA polymerase re-associate and begin the process again

the bacterial transcription process

When the loop is formed, accessibility is lost to certain regions so cleaving occurs

How does the hairpin structure terminate transcription?

nucleolus

RNA polymerase I is located in the _____

nucleoplasm

RNA polymerase II and III are located in the _____

- a cyclic peptide of 8 amino acids

- destroying angel

- used in the analysis of RNA polymerase for transcription ability

- a toxin that stops the transcript process

alpha-amanitin is

very sensitive

RNA polymerase II is _____ to alpha-amanitin

transcription factors

eukaryotic promoter structure has different _______ for different regions

1. One subunit of one TF (transcription factor) binds to promoter and bends the DNA

2. Many other TFs are recruited

3. RNA polymerase binds

4. RNA polymerase C-terminal tail is phosphorylated by a TF and releases an enzyme from the pre-initiation complex, transcription initiation

5. elongation

6. termination

7. pre-mRNA is made

the process of eukaryotic promoter binding

TFIID

recognizes TATA box and other DNA sequences near the start point

TFIIB

recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription

TFIIA

Not required in all promoters; stabilizes binding of TFIID

TFIIF

stabilizes RNA polymerase interaction with TFIIB; helps attract TFIIE and TFIIH

TFIIE

attracts and regulates TFIIH

TFIIH

unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase CTD; releases RNA polymerase from the promoter

when TFs phosphorylate the polymerase C-terminal

the trigger to start RNA synthesis in eukaryote transcription

prokaryotes

in _____ translation begins immediately after enough RNA has exited the RNA polymerase

leave the nucleus to the cytoplasm

pre-mRNA must...

After promoter binding and transcription in eukaryotes

RNA processing occurs...

a 5' cap, 5' UTR, exons, 3' UTR, and a poly-A tail

Mature RNA form

UTR

transcripts present at the ends of RNA

1. snRNPs bind to either end of an intron (U1 and U2)

2. snRNP U1 is replaced with U6 and an active site in created by U2 and U6 coming together

3. The intron is spliced containing the U6-U2 complex

4. The exon junction complex joins exon 1 and 2 to make a mature mRNA

process of splicing in RNA processing

- Weaker splicing signals at alternative splice sites, shorter exon length or higher sequence conservation surrounding orthologous alternative exons influence the exons that are ultimately included in the mature mRNA. This process is mediated by the spliceosome and occurs by exon shuffling, exonization of transposable elements, or constitutively spliced exons.

- Regulation of function

How are specific exons selected in alternative splicing? How is the order of exons decided?

- Alternative splicing is a process controlled by cis-regulatory sequences, which determine whether a gene will be expressed or mRNA will undergo alternative splicing

- Alternative splicing can give rise to different proteins from the same gene, a common strategy to enhance the coding potential of genomes.

How and why does alternative splicing occur? What role does it play?

5' cap

a protein exchange at the ______ in the cytosol serves as an initiation factor for protein synthesis/translation

- control disease state

- cell function differentiation

- regulate protein production

- cancer therapies

- understanding disease mechanism

Why do we want to control gene expression?

stressors and environement

gene expression can be affected by ______ and _____

RNA sequencing

__________ maps out a large number of genes

1. break cell

2. take RNA

3. put RNA through a pipeline with reagents

4. get data

RNA sequencing process

what genes are present/expressed in each tissue

RNA processing allows us to know...

- understand global changes in RNA

- difference between 2 things

purpose of RNA sequencing

neuron subtype (x-axis)

list of genes (y-axis)

how mRNA spectra is organized

microbiome differences

Can be determined with RNA sequencing

housing effect

the microbiome of different organisms (tested with mice) will become the same if they are in the same cage

to prevent undesired changes

why is mRNA destroyed?

siRNA (short interfering RNA, silencing RNA)

how is mRNA destroyed?

1. when and how often a given gene is transcribed

2. splicing process of RNA transcripts

3. selecting which completed mRNAs are exported to the cytosol and where they are localized

4. selecting which mRNAs in the cytoplasm are translated by ribosomes

5. selective destabilization of mRNA in the cytoplasm

6. selectively degrading protein

7. activating, inactivating, or localizing specific protein molecules

What are the 7 steps at which eukaryotic gene expression can be controlled

transcription regulators

proteins that bind using specific amino acids to prevent transcription

- single monomer

- dimer

- heterodimer (2 different monomers bind to make a dimer)

How do transcription regulators bind?

controlled

DNA sequences are replicated in a ___________ manner

mutation

Replication that occurs too rapidly may result in a ___________

one nucleotide change per 10^10 nucleotides each time the DNA is replicated

The mutation rate in humans is about _________________, which is a low level of mutation

cancer

Mutation can lead to _____________ in somatic cells

slowly

DNA replication occurs ____________

Gender (male vs female), evolution, antibodies

Examples of positive mutations (not bad)

constantly, every time you are exposed to something

Anitbodies are mutated ___________

cancer, genetic diseases, neurological diseases

Examples of negative mutations (bad)

3 nucleotides per 10^10 nucleotides per cell division

Ecoli has a mutation rate of _________________

DNA polymerase

______________ is a DNA replication machinery that proofreads and removes errors/mutations in our bodies

To drive the reaction that grows the DNA strand - catalyzing the polymerization reaction, proofreading errors

Why we need DNA polymerase

5' to 3'

_____________ the directionality of the growth of a DNA chain

non-covalent

______ bonds join nucleotide pairs

covalent

______ bonds join the sugar phosphate backbone of DNA

protein, enzyme

DNA polymerase is a ________

catalyst

How an enzyme acts

1. A nucleoside triphosphate pairs with a base in the template strand

2. DNA polymerase catalyzes a covalent linkage of nucleoside monophosphate to by releasing a diphosphate from the nucleoside triphosphate

3. The strand grows

How DNA polymerase adds new nucleotide pairs to DNA

mutation

Changes in nucleotides

1. DNA polymerase binds and begins synthesis in the 5' > 3' direction

2.1 The polymerase adds an incorrect nucleotide

2.2 The mis-paired nucleotide is removed via proofreading

3. The correct nucleotide is added

4. Synthesis continues

How DNA polymerase proofreads bases added to the DNA

base pair interactions

Incorrect additions are determined by analyzing ___________

E (editing)

Proofreading on the DNA polymerase occurs in the ______ site

P

Polymerization on the DNA polymerase occurs in the ______ site

synthesis of new DNA strands

Proofreading with DNA polymerase only occurs during _______

AT rich region

An indicator that there is a replication origin

two

There are _________ replication forks

two

In DNA replication there are _____ template strands

The DNA strands are antiparallel and replication runs in the 5' to 3' direction

Why DNA replication runs in 2 different directions

Draw the DNA replication process

It helps bring primers in order to start DNA synthesis

Why we need DNA primase

primers

Primase is an enzyme that synthesizes short RNA sequences called _______, which are the starting point for DNA synthesis

large

The replication region is a __________ region, which contains the AT rich sequence

RNA polymerase

DNA primase is a type of __________ because it produces RNA molecules

DNA replication stops

What happens if RNA primase stops being produced