Molecular Bio test 1

What are the properties of bacterial RNA Polymerase?

Made up of 5 subunits: Alpha (to fo them), Beta, Beta Prime, and Omega

What is transcription? What is the responsible enzyme?

The synthesis of RNA using DNA as a template. RNA Polymerase is responsible.

What is the function of the alpha subunit?

DNA-Binding site

What is the function of the Beta Subunits?

DNA-Binding site

What is the function of the Beta Prime Subunits?

DNA-Binding site

What is the function of the Omega Subunits?

RNA Polymerase assembly and stability

What are bacterial promoters?

sequences necessary to promote transcription via RNA polymerase.

Where are promoters located

upstream or 5’ of genes

How is a promoters strength determined?

it depends on the match of the sequence to the consensus bases; the more bases the match to the consensus, the stronger the promoter can bind better

What are the consensus bases?

The most common base in each position.

What happens if you change the bases in a sequence?

The bacterial promoter activity will decrease because they cannot bind.

How can DNA Binding proteins such as RNA polymerase be produced in vitro studies?

by generating recombinant proteins in E. coli.

How to tag a protein with GST

determine if the tag adds to the C- or N-terminal by adding some to both, then add GST to the gene of interest and remove the stop codon. Add the protein to a the plasmid. Let transcription and translation occur.

What are the pros of using recombinant proteins versus native proteins in research?

Can make a lot of protein, you can tag a protein to track it, can help a protein fold and become soluble, can bind an antibody to a tag for blots, oyu can add mutations to plasmid without using the original cell as a source.

What are the cons of using recombinant proteins versus native proteins in research?

If the proteins don’t fold, they fall out of solution. Proteins may not bind. Proteins may not be phosphorylated.

What are the molecular requirements for transcription?

RNA polymerase, NTPs, Mg2+ and a DNA template

What is the transcription reaction?

A new phosphodiester bond is formed between old sugar and new, incoming sugar in the 5’ to 3’ direction.

What are the NTPs in transcription? What do they do?

Nucleotides and they are the bases with 3 phosphate groups used in building the RNA strand

What is Mg2+ used for in transcription

They have a positive charge where everything else has a negative charge, so they are used to make the hydroxy group attack phosphate group to make phosphodiester bond.

What requirements are there for a DNA strand to be a template?

They must have a promoter for the RNA polymerase to bond to.

Where does transcription start?

At the +1 region on the Template strand. RNA polymerase only binds to one strand of DNA to make the exact copy of a coding strand.

What is the UTR in RNA?

Untranslated region that does not et translated into a protein.

How are UTRs formed?

Rna doesn’t have start or stop codons, and RNA polymerase only starts at the promoters, there are regions of RNA that are before DNA start codons and after stop codons that are transcribed. These are sections that do not get translated into proteins.

What are the molecular events that define transcription initiation?

RNA Polymerase holoenzyme binds to the DNA, then some of the DNA is unwound and transcription is initiated and continues until the terminator sequence is reached.

How does RNA polymerase bind to DNA?

With the sigma factor, which changes the shape of RNA polymerase. Sigma also binds to the -35 and -10 regions of the DNA.

How is the open complex formed?

with RNA polymerase either binding to sigma or beta prime subunits. It can only happen with there’s both the core enzyme and either sigma or beta prime.

What is abortive initiation?

When short bands of RNA are produced by RNA polymerase before transcription occurs completely. Then after a few short strands are made, something changes that allows for complete transcription to occur.

What is the function of abortive initiation? (theories, they don’t know)

test mechanism to check if everything is working correctly, regulation, or can maybe control gene expression

What are the molecular events that define transcription elongation?

RNA polymerase moves along the DNA chain to make a growing chain of RNA

What happens to sigma during transcription?

It is released from RNA polymerase. This happens so sigma can reassociate with RNA polymerase somehwere else.

What are the molecular events that define transcription termination?

RNA polymerase reaches the info needed for termination down stream of the stop codon in DNA (3’ UTR)

What happens with a rho independent terminator?

a stem and loop is produced in the RNA making it, double stranded before there is a run of the same nucleotides, like U’s. There are weak base pairs between the U in RNA and the A in Dna, so RNA polymerase falls off

What happens with a rho dependent terminator?

rho binds to rut site then can move left to right. eventually it catches up with RNA polymerase and removes it from template strand

Why does transcription termination matter?

Without it bacteria cells can waste enough materials with transcription and end up dying. They also carry virus genomes, so if transcription occurs there then the virus is activated.

What is the sequence reaction in next gen sequencing

Copying a strand of DNA but you add fluorescent bases that block transcription, so when it stops, the color associated with the base is present and recorded

how many eukaryotic RNA polymerases are found in each cell?

3- RNA polymerase 1, RNA polymerase 2, RNA Polymerase 3

what does RNA polymerase 1 do in Eukaryotic cells?

transcribe rRNA

what does RNA polymerase 2 do in Eukaryotic cells?

transcribe mRNA and small regulatory RNAs

what does RNA polymerase 3 do in Eukaryotic cells?

transcribe tRNA, 5S RNA, and small nuclear RNA

How does the eukaryotic RNA polymerase repertoire compare with the bacterial RNA polymerase repertoire?

Eukaryotic cells contain 3 different RNA polymerases that are highly specialized and bacterial cells rely on a single RNA polymerase

What are the characteristics of RNA polymerase 2

they have multiple subunits. the c-terminal domain is rich in tyrosine, serine and threonine residues

What are the characteristics of the eukaryotic promoters?

The BRE, TATA box, INR, DPE regions

What does BRE do?

helps position the transcription machinery correctly

What does TATA box do?

establish where transcription starts

What does INR do?

Initiator element at the +1 region, where RNA polymerase starts making RNA

What does DPE do?

downstream promoter element- it is located downstream if the transcription start sight to help recruit or position transcription machinery.

What is the TBP

Tata-binding protein. recognizes the TATA box

What is the TAFS

TBP-associated factors. they help recognize promoter elements

What is the TFIID

major transcription-factor complex that is made up of TBP and TAFS. it recognizes promoter attachments.

What is the TFIIB

general transcription factor that binds near the BRE region

What are the molecular events that define transcription initiation, elongation and termination?

Polymerase 2 is recruited to DNA, the transcription bubble forms by TFIIH unwinding DNA, CTD is phosphorylated during initiation, elongation, dephosphorylation of CTD, transcription is terminated.

How does RNA Polymerase 2 find the promoter

general transcription factors

What are the requirements of a transcription reaction set up?

RNA polymerase, buffer, NTPs, DNA template, gel electrophoresis

How are the 3 eukaryotic RNA polymerases purified? (includes naming)

By ion-exchange chromatography. RNA polymerase comes off at low salt concentrations, RNA polymerase 2 comes off at intermediate salt concentrations and RNA polymerase 3 comes off at high salt concentrations.

What subunits does pol 2 have?

12 subunits (RPB1 through RPB12), with a repeating heptapeptide sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser.

What is the RNA polymerase 2 C-Terminal domain (CTD)

A region rich in Tyrosine, serine and threonine which act as a coordinator for phosphorylation and co-transcriptional processing.

What are the RNA polymerase inhibitors

sigma-amanitin and actinomycin D

What does sigma-amanitin do

binds to RNA polymerase and changes the structure, preventing the ability to transcribe. Mainly affects Pol 1 and 2

what does actinomycin D do

Wedges itself in the minor groove of double stranded DNA, blocking all RNA polymerases

How does Pol 2 assemble at the promoter

1. TFIID: Lands first. Its TBP subunit grips the TATA box and bends the DNA.

2. TFIIB: Lands next. It measures the distance to the start site and sets the direction.

3. Pol II + TFIIF: TFIIF acts as a chaperone, carrying RNA Pol II directly to the promoter.

4. TFIIE: Joins the group to recruit and turn on the final factor.

5. TFIIH: Lands last. It unzips the DNA (Helicase) and adds phosphates to the Pol II tail (Kinase) so the polymerase can break free and start zooming down the gene.

What does TFIIH do?

unzip DNA by melting at the start site, then phosphorylates serine 5, making polymerase start transcribing.

steps of transcription

• Step 1: Initiation (Start): Serine 5 gets phosphorylated by TFIIH. This releases the polymerase from the starting line and recruits the capping machinery.

• Step 2: Elongation (Build): Serine 2 gets phosphorylated by P-TEFb (while Serine 5 drops off). This keeps the polymerase moving and recruits the splicing machinery.

• Step 3: Termination (Finish): The heavy Serine 2 signal recruits the tailing (poly-A) machinery to finish the RNA. All phosphates are then stripped off to reset the polymerase for the next gene.

What is the torpedo mechanism for eukaryotic transcription termination

RNA Pol2 transcribes a signal that recruits an endonuclease enzyme to the serine 2 tail of polymerase, and cuts the RNA in two. The front half is the pre-mRNA and the back half is eaten by the exonuclease, which travels down the sequence, colliding with the RNA pol and forcing it to fall off.

What does endonuclease do?

It is the RNA-cutting enzyme used in termination

What does exonuclease do?

It is the RNA-degradation enzyme used in termination

what is the RNA polymerase core enzyme

the main enzyme made of alpha, alpha, beta, betaprime and omega subunits

what is the holoenzyme

core enzyme plus sigma

Structure and function of sigma 70

built to recognize the -10 and -35 promoter regions of DNA for transcription and its job is to bind to RNA polymerase and recognize the promoter, then melt the DNA thats being transcribed

structure and function of sigma S

recognizes the -10 promoter region and melts DNA< but has low affinity to -35 region.

What is the difference between KMnO4 footprinting and DNase footprinting?

• DNase I footprinting → detects where proteins bind and protect DNA from cleavage.

• KMnO₄ footprinting → detects where DNA is unwound or distorted by protein binding.

What does adding competitors to EMSA or filter-binding assays show?

whether the protein-nucleic acid interaction is highly specific or just random, non-specific background binding depending on a decrease or consistant binding signal

What is a recombinant protein?

A protein made by putting a gene from one organism into a host cell (like bacteria) so the cell can mass-produce it.

Why make recombinant proteins instead of taking them from nature?

It is safer, cheaper, and faster than harvesting from animals or humans. It gives you a large, pure supply.

What are the 3 main phases to make them?

• Copy & Paste: Put the target gene into a DNA vehicle (vector).

• Grow: Put the vector into cells and tell them to make the protein.

• Clean: Break open the cells and purify the target protein

How do you know if the process worked?

• Size Check (SDS-PAGE): Shows a single line at the right weight to prove it is pure.

• Activity Test: Proves the protein actually works

Why use a 6xHis tag?

rarely changes the protein's shape. It is perfect when you need the protein to stay as close to its natural form as possible.

Why use a GST tag

It increases solubility and prevents the protein from aggregating.

What is the purpose of in vitro transcription?

It synthesizes RNA from a DNA template in a cell-free system using an RNA polymerase.

Why is in vitro transcription used?

It generates large amounts of specific RNA molecules for structural studies, hybridization probes, and translation assays.

What is the purpose of primer extension analysis?

It maps the exact transcription start site of a specific RNA and quantifies the amount of that transcript.

How does primer extension work?

A labeled primer binds to a specific RNA molecule, and reverse transcriptase extends it until it reaches the five prime end of the RNA template.

How are the results of primer extension analyzed?

The extended DNA products are separated on a gel alongside a sequencing ladder to determine the exact nucleotide where transcription began.