molecular bio exam 3

Central Dogma of molecular biology

DNA ->via transcription to RNA -> via translation to protein

mRNA

carries coding info for proteins

tRNA

brings amino acids to the ribosome

rRNA

catalyzes protein synthesis, makes up 80% of RNA.

Effect of DNA errors on proteins

affect RNA transcribed from it, results in faulty or nonfunctional proteins.

RNA

Ribonucleic acid, a single-stranded molecule involved in various roles such as coding, decoding, regulation, and expression of genes.

Difference between RNA and DNA bases

RNA uses uracil (U) instead of thymine (T).

Function of RNA polymerase

synthesizes RNA by reading the DNA template strand during transcription.

Stability of RNA

has a 2' OH group, making it more chemically reactive and prone to degradation.

Stability of DNA

lacks 2' OH group, is double-stranded, protecting bases from degradation.

Structural property of RNA

single-stranded, can fold back on itself and form complex secondary structures.

Genome

The complete set of genetic material (DNA) in an organism.

Transcriptome

The full set of RNA transcripts present in a cell at a given time.

Steps of transcription

1. RNA polymerase binds to DNA. 2. DNA strands separate. 3. RNA polymerase adds nucleotides. 4. RNA exits, DNA re-anneals.

Template strand for transcription

(non-coding strand) is read by RNA polymerase to make RNA.

Coding strand

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

Transcription bubble

unwound segment of DNA where RNA polymerase is actively transcribing RNA.

number of RNA polymerases in bacteria

One

number of RNA polymerases in eukaryotes

Three

Eukaryotic RNA polymerase similar to bacterial RNA polymerase

RNA polymerase II.

Sigma factor

bacterial protein, helps RNA polymerase recognize promoter region of a gene.

Function of sigma factor

It binds to the promoter to position RNA polymerase correctly and determines which genes are transcribed.

Functions of different sigma factors

σ70: housekeeping genes, σ38: starvation genes, σ32: heat shock, σ54: nitrogen uptake.

RNA holoenzyme

A complex of RNA polymerase and a sigma factor.

Promoters

DNA sequences that signal where transcription should begin.

Consensus sequence in a bacterial promoter

A conserved sequence at -35 and -10 regions where sigma factors bind.

UP element

sequence upstream of promoter, enhances transcription by binding alpha subunit of RNA polymerase.

DNA footprinting assay

A technique used to study protein-DNA interactions.

Footprinting assay

A technique used to identify where proteins bind on DNA by observing protected regions after DNase digestion.

How does the footprinting assay work?

DNase I randomly cleaves unprotected DNA; protein-bound areas are not cleaved and appear as gaps on a gel.

Bacterial transcription initiation

RNA polymerase binds promoter, melts DNA, forms open complex, starts RNA synthesis.

Abortive initiation

RNA polymerase starts and stops repeatedly without leaving the promoter, producing short RNA fragments.

Promoter clearance

When RNA polymerase successfully escapes the promoter and continues transcription after >10 bp.

Mechanisms of transcription termination in bacteria

Rho-independent and Rho-dependent termination.

Rho-independent termination

A hairpin forms in the RNA followed by U-rich region, causing polymerase to fall off.

Rho-dependent termination

Rho helicase binds to rut site on RNA and chases polymerase, using ATP to unwind RNA and end transcription.

Channels in RNA polymerase

DNA entry channel, NTP entry channel, RNA exit channel.

Pin region of RNA polymerase

Stabilizes the open state of the DNA by holding DNA strands open

Types of proofreading in transcription

Kinetic proofreading and nucleolytic proofreading.

Kinetic proofreading

Polymerase reverses the phosphodiester bond reaction if a mismatch is detected.

Nucleolytic proofreading

RNA polymerase backtracks, and nuclease activity cleaves mismatched RNA.

Eukaryotic RNA pol II complexity

has more subunits, a C-terminal domain (CTD) involved in regulation.

CTD (c terminal domain) of RNA pol II

A flexible tail that acts as a docking site for transcription and RNA processing factors.

TBP

TATA-binding protein, essential for transcription initiation at most eukaryotic promoters.

TATA box location

About -30 bp upstream of the transcription start site.

RNA polymerase I function

Transcribes most rRNAs, about 80% of total transcription.

RNA polymerase II function

Transcribes mRNA and some snRNAs.

RNA polymerase III function

Transcribes tRNAs, 5S rRNA, and some small RNAs.

Pol III promoters uniqueness

located within gene, require TFIIIB and TFIIIC.

Torpedo model of transcription termination in eukaryotes

CPSF cleaves RNA at AAUAAA, then Xrn2 exonuclease degrades remaining RNA and dislodges polymerase.

Northern blot

Used to detect RNA levels and size using radiolabeled probes.

RT-PCR

Reverse transcription PCR: converts RNA to cDNA and amplifies it.

qRT-PCR

Quantitative PCR using cDNA to measure transcript levels with fluorescent signals.

RNA sequencing

converting to cDNA and sequencing. Shows global expression.

Gel electrophoresis

Separates molecules by size; smaller molecules migrate faster through agarose gel.

Probe in hybridization

A labeled DNA or RNA fragment that binds to its complementary sequence to detect specific molecules.

pre-mRNA

The immediate product of transcription before processing.

What is the coding strand in transcription?

DNA strand w/ same sequence as RNA transcript, but 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

fully processed transcript, 5' cap, poly(A) tail, 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

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

1 gene produces 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

by 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)

complexes at exon-exon junctions, determine pre-mRNA from mRNA.

EJCs in RNA export

guide export machinery to properly splice 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.