Molecular Cell Biology Test 2

How does systemic lupus erythematosus affect the body?

causes the body to produce antibodies to multiple vital proteins (such as PCNA), disrupting their function and damaging cell function

symptoms of systemic lupus erythematosus

inflammation throughout body, tissue damage, fatigue, frequent fevers, joint pain

what testing system is used for diagnosis of systemic lupus erythematosus?

ELISA

What does ELISA stand for

enzyme linked immunosorbent assay

How does ELISA work?

• create grid of wells, each lined with different protein commonly attacked by the lupus

• sample blood of patient, place into wells

• If the blood carries antibodies for that protein, it will stick to the walls

• Use secondary antibody for protein antibody

• Secondary protein carries HRP (horseradish peroxidase) which will convert TMB into a blue coloured substrate

• Therefore, if blue appears in the well, then there were antibodies for that protein present

Who was the leading voice for the Central Dogma theory?

Francis Crick

Transcription expresses…

genes

What direction does RNA polymerase read template strand?

3’-5’

What direction does RNA polymerase synthesize RNA?

5’-3’

Which strand of DNA is the template strand?

The strand read by RNA polymerase to synthesize a complimentary strand of RNA

Which strand of DNA is the coding strand?

The strand NOT read by RNA polymerase, instead it matches up base-wise with the synthesized RNA strand

the strand of mRNA produced is base-wise the same as the….

coding strand

What is different between DNA nucleotide and RNA nucleotide

• They have different sugars (ribose for RNA, deoxyribose for DNA)

• They have 1 different base each (DNA = Thymine, RNA = Uracil)

What sugar is this?

Ribose

What base is this?

Deoxyribose

What base is this?

Uracil

What base is this?

Thymine

What is the purpose of mRNA

codes for proteins

what percentage of all RNA in a cell is mRNA

2%

What is the purpose of rRNA

acts as the RNA component of ribosomes, never translated

what is the purpose of tRNA

acts as an adapter between mRNA and protein formation, carries AAs

who theorized existence of tRNA before it was actually discovered

Francis Crick

what is the purpose of miRNA

controls the expression of genes

size of miRNA

<22 bases in length

What is the purpose of lncRNA

regulates gene expression

what is the purpose of other types of RNA found in cells

also aids in expression of genes

how long is lncRNA

200+ bases

RNA polymerase is ____-dependant

DNA

does RNA polymerase require helicase to open the helix?

No, it does not

what is the function of RNA polymerase

binds to DNA, opens up double helix, begins synthesis by reading template strand

RNA polymerase acts as both a _____

catalyst and denaturing agent

Types of RNA polymerase

RNA poly I

RNA poly II

RNA poly III

Purpose of RNA poly I

transcribes and initiates rRNA transcript

Purpose of RNA poly II

transcribes protein coding transcripts (mRNA) and miRNA

purpose of RNA poly III

transcribes tRNA transcripts and some rRNA

What is the promoter sequence of DNA

region which is never transcribed, acts as site where RNA poly can bind and initiate transcription

What number is assigned to the site of initiation of transcription

+1

where are the promoter sequences in prokaryotes?

-35 (TTGA box) and -10 (TATA box)

Sequence of nucleotides at -35 (P)

TTGACA

Sequence of nucleotides at -10 (P)

TATAAT

Are the promoter sequences the same for every organism?

No, but the ones used are the most common and applicable sequences

What allows RNA poly to bind to DNA in prokaryotes

sigma factor protein

how does the sigma factor protein work? (P)

acts as guide for RNA poly to find where to start transcription and bind to DNA

How does transcription initiation in Eukaryotes differ from P?

multiple proteins bind to TATA box to encourage transcription

general transcription factors

Used by all genes being transcribed, no specificity

Specific transcription factors

used by specialized cells to produce certain proteins, uses specific factors which bind to pull GTFs in

Types of GFPs

• TFIID → TBP, TAF

• TFIIB

• TFIIF

• TFIIE

• TFIIH

Purpose of TFIID factor

first to bind to DNA, causes distortion that allows TFIIB to bind

Purpose of TBP factor

binds to TATA box,

Purpose of TAF factor

associates with TBP at TATA box, helps in initiation

Purpose of TFIIB factor

binds to DNA after TFIID to bring RNA pol to it

Purpose of TFIIF factor

assists in binding RNA poly to promoter site

TFIIE factor purpose

aids in forming initiation complex and in elongation process

TFIIH factor purpose

opens double helix at start site, also phosphorylates RNA pol II to release all other TFs and allow it to bind to DNA

what sequence is found at -35 (E)?

G/C G/C G/A CGCC (TFIIB)

what sequence is found at -10 (E)?

T A T A A T A A/T, TFIID subunit

what is an example of a specific transcription factor

MYoD

How does MYoD work?

acts as regulatory protein, binds to Ebox sequence due to high affinity near promoter

Ebox sequence

CANNTG

MYoD is ______

muscle-specific

MyoD is a _____ transcription regulator

Master

MyoD is expressed inly in ______ cells

muscle

the Ebox is found in

muscles cells, is specific

what would be the conseqence of no MyoD in an individual

no muscle cells would be produced, whih would result in no muscular tissue within that individual

what happens to cells who do not normally express MyoD when they are forced to express it

they transition into muscle cells → they begin producing muscular proteins and sequences, which transforms the cell

What is the phenomenon called when a cell is forced to express a different phenotype than expected or originally coded for (ex. forced MyoD expression in a non-muscle cell)

Transdifferentiation

how can a cell be forced into Transdifferentiation using MyoD?

attaching a sequence that codes for MyoD with a strong promoter that is recognized by the cells, such as CMV (viral promoter)

Transdifferentaition into neural cells

Done using NeuroD, will transform cell into neuron using overexpression of particular proteins

How would you determine a stretch of DNA is part of a promoter without knowing sequence of promoter?

• know that promoter is upstream of gene sequence

• use sequence for GFP to test immediately of gene is working

• being damaging suspected region of bases, test if GFP is produced

• Once part is damaged and no GFP is produced, the promoter region can be identified

Promoter bashing

damaging suspected promoter regions to identify where specifically boxes lay

truncation

process in promoter bashing where end sequences are chopped off to see if transcription is inhibited or not

nucleic acid hybridization

specific transcript identification process that uses labeled mRNA probes complimentary to target to identify

How are RNA probes made for hybridization?

• all RNA is isolated in organism

• RNA is converted into cDNA using reverse transcriptase; cDNA is amplified using PCR to produce multiple double stranded copies

• T7 promoter is added, T7 RNA Polymerase binds to it and produces ssRNA → fluorochrome or gold particle is added to produced RNA for detection

using nucleic acid hybridization and northern blotting

process involving production of cDNA using reverse transcriptase to bind to mRNA, cDNA is then electrophoresed and northern blotted → used to identify quantities of RNA types present in an organism

does reverse transcriptase need a primer?

yes

what is PCR

polymerase chain reaction, allows for large volume of replication to occur

what is pcna

Proliferating cell nuclear antigen → type of protein

is DNA or RNA more stable?

DNA is more stable

Northern blotting

process of electrophoresis on RNA molecules to separate by size, which are blotted onto a membrane sheet → into saline solution containing RNA probes, which hybridize with matching strands

Then, glowing is tested for and the target sequences are identified and excised from the paper

is northern blotting sensitive?

it is moderately sensitive, and needs lots of material or the results will not be as accurate

what does RT-PCR stand for

reverse transcription based polymerase chain reaction

what is RT PCR

• start with RNA

• Reverse transcribe into cDNA with primer

• use PCR to amplify cDNA, which can then be electrophoresed

is RT PCR sensitive

it is extremely sensitive; any changes get amplified

densiometery

using band thickness from electrophoresis to determine quantity

what does ISH stand for

In situ hybridization

what is ISH

way of identifying locations of mRNA produciton inside tissue

How does ISH work

use RNA probes which match with specific mRNA inside organism and wash across tissue → test visualization and detect probes in bonded in tissue

What are the three main kinds of modifications done to mRNA in eukaryotes

5’ capping, 3’ poly a tail addition, RNA splicing

what is 5’ capping

the addition of methyl guanosine cap to 5’ end of mRNA, protects molecule from degradation and helps in recognition of molecule

Does ISH have good resolution

It has a high resolution and is very quick

what is 3’ polyadenylation

addition of 50-200 A bases to 3’ end of mRNA, prevents RNA from degradation and increases survival

what enzyme adds adenine to 3’ tail

polyA-polymerase

does poly A polymerase need a template?

No, as it does not need to read one to add the tail

What is the name of the untranslated regions at the 5’ and 3’ ends of mRNA

5’ UTR and 3’ UTR

when does RNA processing occur

During transcription

what enzyme does the RNA processing

RNA polymerase II

how does RNA processing occur during transcription

C terminal tail of RNA poly II carries enzymes that signal for modification to occur when released, also carries phosphate groups to phosphorylate to allow enzymes to bind

How to determine consensus sequences that represent splice sites using bioinformatics

• lay out sequence of full DNA, then sequence of spliced DNA (mRNA is published as DNA sequence)

• Remove matching segments, left with introns

• Find similarities between starting/ending sequences

What are the consensus sequences for intron splicing

GUXXX at 5’ end, AG at 3’ end. However, it is more complicated than just these bases (these can occur in exons and not cause splicing)

what base does Y refer to in sequences

any pyrimidine