Molecular Bio Exam 3

pre-mRNA

The immediate product of transcription before processing.

What is the coding strand in transcription?

The DNA strand that has the same sequence as the RNA transcript, except it has T instead of U.

What is the non-coding (template) strand in transcription?

The DNA strand used as a template by RNA polymerase; it is complementary to the RNA transcript.

How does the RNA transcript relate to the coding and template strands?

RNA matches the coding strand, but has U instead of T, and is complementary to the template strand.

How do you identify the RNA transcript from the template strand?

take the complement of the template and replace T with U to reflect RNA.

How do you identify the coding strand when given a template strand?

Take the complement of the template and switch the direction (5′ to 3′); that’s your coding strand.

mature mRNA

A fully processed transcript that includes a 5' cap, poly(A) tail, and no introns.

steps of eukaryotic mRNA processing

1. 5' capping, 2. Poly(A) tail addition, 3. Splicing, 4. Nuclear export.

5' cap on mRNA

It protects the mRNA from 5' to 3' exonucleases and is required for translation.

structure of the 5' cap

7-methylguanosine added backwards to the 5' phosphate of the transcript, methylated at the 7 position.

enzyme that adds the 5' cap

Guanylyltransferase binds to the CTD of RNA Pol II and caps the mRNA as it emerges.

CBC

Cap-binding complex; it binds to the 5' cap and helps tether the mRNA to Pol II.

necessity of 5' capping for translation

Shown by using radioactive labeling and density gradient centrifugation to compare transcripts with and without a 5' cap.

poly(A) tail

A long stretch of adenosines added to the 3' end of the mRNA.

functions of the poly(A) tail

Protects the mRNA from degradation and helps recruit ribosomes for translation.

enzyme that adds the poly(A) tail

Polyadenylate polymerase (PAP).

sequence that signals cleavage during polyadenylation

The AAUAAA consensus sequence.

protein that binds the poly(A) tail

Poly(A) binding protein (PABP).

exons

Sequences that remain in the final mRNA and are translated into protein.

introns

Non-coding sequences between exons that are removed during splicing.

function of the 5' UTR

Contains regulatory sequences that help initiate translation.

3' UTR

Contains regulatory sequences and the polyadenylation site.

alternative splicing

A process that allows one gene to produce multiple mRNA isoforms by skipping or including different exons.

poly(A) site choice

Use of different cleavage and polyadenylation sites to generate different mRNA transcripts from the same gene.

splicing machinery recognition of introns

Through conserved sequences in introns: 5' splice site (GU), branch site (A), and 3' splice site (AG).

branch point in splicing

An internal adenosine nucleotide upstream of the 3' splice site that participates in lariat formation.

function of RNA export

Transports mature mRNA from the nucleus to the cytoplasm where translation occurs.

protein that recruits TREX to Pol II

TREX is recruited to Pol II to help in RNA export.

exon junction complexes (EJCs)

Protein complexes deposited at exon-exon junctions during splicing, used to distinguish pre-mRNA from mRNA.

EJCs in RNA export

They guide the export machinery to properly spliced transcripts.

exportins

Proteins that bind RNA and transport it through the nuclear pore complex.

mRNA after translation

It is degraded and recycled.

first removal during RNA degradation

The 5' cap and the poly(A) tail are removed first.

exonucleases

Enzymes that degrade RNA from the ends.

exosome

A complex of 10 ribonucleases that degrade mRNA.

P bodies

Processing bodies that store or degrade mRNAs.

transport receptor Ran

It transports noncoding RNAs to the cytoplasm when bound to GTP.

trans-acting factors

Proteins that interact with DNA to regulate transcription.

cis-acting elements

DNA sequences like promoters or enhancers that regulate transcription of nearby genes.

influence of trans-acting factors on transcription

They bind DNA directly or are recruited by other proteins to activate or repress transcription.

examples of cis-acting elements

Promoters, enhancers, silencers, activator/repressor binding sites.

testing mutation in cis or trans

Introduce a plasmid with a normal copy of the gene. If expression is restored, the defect is trans.

lac operon

It helps E. coli digest lactose when glucose is not available.

main genes in the lac operon

lacZ, lacY, lacA - involved in lactose metabolism.

role of the lac repressor

It binds the operator and blocks RNA polymerase when lactose is absent.

effect of lactose presence

Allolactose binds the repressor, inactivating it, allowing transcription.

CRP protein

cAMP receptor protein that activates transcription when glucose is low and cAMP is high.

helix-turn-helix motif

A structural motif where one helix binds DNA, stabilized by a turn and another helix.

leucine zipper

Two helices dimerize through leucines and bind DNA, though the zipper region itself doesn't bind DNA.

zinc fingers

Motifs stabilized by zinc ions that allow proteins to bind DNA in the major groove.

heterochromatin

Tightly packed chromatin that is transcriptionally silent.

euchromatin

Loosely packed chromatin that is transcriptionally active.

nucleosome repositioning

Moves nucleosomes to expose or hide DNA regulatory elements.

histone acetylation

Addition of acetyl groups by HATs, loosening chromatin to activate transcription.

histone deacetylation

Removal of acetyl groups by HDACs, tightening chromatin to repress transcription.

DNA methylation

Addition of methyl groups to CpG sites, repressing transcription by blocking transcription factor binding.

siRNA

Synthetic or viral RNA that forms perfect matches with mRNA to trigger degradation.

miRNA

Endogenous RNA that regulates gene expression, usually by blocking translation.

pri-miRNA transcription location

In the nucleus.

Drosha

Cuts pri-miRNA into pre-miRNA.

fate of pre-miRNA after Drosha cleavage

It is exported to the cytoplasm and enters the RISC complex.

Dicer

Processes pre-miRNA into a double-stranded miRNA duplex.

RISC

Uses one strand of miRNA to guide it to target mRNAs for degradation or translational repression.

Which of the following does NOT occur in prokaryotic transcription?

5′ Cap attaches to CTD

Prokaryotic Transcription

Bacteria making RNA from DNA

What are the three types of RNA made during transcription?

mRNA, rRNA, tRNA

What happens during the initiation stage of transcription?

RNA polymerase binds the promoter and begins RNA synthesis.

What happens during elongation in transcription?

RNA polymerase moves along the DNA and builds RNA in the 5′ → 3′ direction.

What happens during termination of transcription?

RNA polymerase stops, and the RNA strand is released.

Where does transcription happen in prokaryotes?

In the cytoplasm (because there’s no nucleus).

Where does transcription happen in eukaryotes?

In the nucleus.

Do prokaryotes process their mRNA (splicing, 5′ cap, poly-A tail)?

No — their RNA is usually ready to be translated right after it's made.

Do eukaryotes modify their mRNA before translation?

Yes — they add a 5′ cap, a poly-A tail, and remove introns (splicing).

What helps RNA polymerase bind the promoter in prokaryotes?

The sigma factor.

What helps RNA polymerase bind in eukaryotes?

General transcription factors and other proteins.

Can transcription and translation happen at the same time in prokaryotes?

Yes — they are coupled since both happen in the cytoplasm.

Can transcription and translation happen at the same time in eukaryotes?

No — transcription happens in the nucleus, translation in the cytoplasm.

What helps terminate transcription in prokaryotes?

Rho factor or hairpin loop structures (Rho-independent).

What helps terminate transcription in eukaryotes?

Different signals depending on the polymerase; often includes cleavage and polyadenylation.

are the three main stages of transcription the same or different for eukaroytes and prokaryotes

same, just have different details

What is a DNA footprinting assay used for?

To determine where a protein binds to DNA.

How does a DNA footprinting assay work?

DNA is incubated with a protein, then treated with nuclease. The protein blocks cuts where it’s bound, creating a protected region.

What does a “footprint” look like on a gel?

A gap in the ladder of bands where DNA wasn’t cut because it was protected by the protein.

What does it mean if Protein A creates a larger footprint than Protein B?

Protein A binds a larger region of DNA — it protects more bases from cleavage.

What happens to DNA regions not protected by protein in a footprinting assay?

They are cleaved by the nuclease and show up as bands on the gel.

Pol I makes rRNA →

leaves the nucleus to become part of ribosomes

Pol II makes mRNA →

this stays in the nucleus first for processing (cap, tail, splicing)

pol III makes tRNA (and some small RNAs) →

these are used in translation, so they leave the nucleus

Pol I and Pol III make RNAs that go

to the cytoplasm

Pol II’s product (mRNA)

doesn’t leave until it’s processed

AAUAAA

poly(A) addition site, signals where the poly-A tail should be added at the 3′ end of eukaryotic mRNA

GU......AG

Splice site, GU is the 5′ splice donor and AG is the 3′ splice acceptor,

where is GU......AG found

at intron/exon boundaries

TATAAA

TATA binding site (TATA box), part of promoter region in eukaryotes

TATA binding site recognized by

TFIID

TFIID

general transcription factor in eukaryotic transcription, first to bind promoter region, starts transcription initiation.

RNA Hairpin followed by UUU

Rho-independent termination, in prokaryotes causes RNA polymerase to fall off the DNA

Why is double-stranded RNA better at gene silencing?

It loads onto the RISC complex

dsRNA gets processed by

Dicer into siRNA or miRNA

What is RNA interference (RNAi)?

A gene-silencing mechanism in eukaryotes that uses small RNAs to block or degrade target mRNA.