BIMM 100 Midterm 2

Primer

DNA or RNA complementary sequence that attaches to template DNA (acting as a starting point)

Allows DNA polymerase to add nucleotides/not required in Transcription

Polymerase Chain Reaction (PCR)

Purpose is to copy and amplify specific region of double strand DNA

Dependent on DNA polymerization in a test tube, single strand template, primer, dNTPs.

Simple sequence repeats

Non-functional

Location of repeats in genome are fixed (same location in genome for all humans)

Number of repeats at each location are variable among individuals

How to amplify Simple sequence repeats

Through PCR and Electrophoresis

Application for Paternity and Criminal cases

Reverse transcription

Scenario: RetroVirus infects human and will inject viral RNA

1. Complementary DNA strand generated from viral RNA (RNA dependent and DNA polymerase),

2. viral RNA is digested/only DNA in cell (RNA nuclease),

3. cell will make double stranded DNA (DNA dependent and DNA polymerase),

4. DNA integrated into human genome

This is possible due to enzyme Reverse transcriptase (single strand of RNA into double strand DNA)

Step 4 uses enzyme Integrase

What happens once viral RNA to DNA is inserted into human genome?

Is silenced, however double stranded DNA will be transcribed back into RNA and will be translated to create more enzymes (Reverse transcriptase, Integrase, Protein coat, Protease)

Do you need a primer for DNA polymerization or RNA polymerization (transcription)?

For any DNA polymerization you do, however RNA polymerization you don’t

Reverse transcription in Eukaryotes

DNA fragments called mobile elements (DNA fragments move within and between chromosome location) in Eukaryotic cells

Mobile elements considered non-functional

Types of Mobile elements

DNA transposons (Cut and paste) Original site loses DNA material

Retro transposons (Copy and paste) Original site maintains DNA material since DNA fragment uses RNA intermediate (undergoes reverse transcription)

Mechanism for DNA transposon

DNA site undergoes transcription generating RNA, translated into enzyme called Transposase (cuts out DNA transposon site and integrates to target region)

Mechanism for Retrotransposition

Retrotransposon DNA transcribed to RNA, translated into protein Reverse transcriptase, this enzyme will reverse transcribe RNA into double stranded DNA, Integrase inserts retrotransposon DNA to target region

Autonomous transposition

Mobile DNA element encodes the enzymes needed for transposition

Although have genetic material, they need host gene expression machinery to express these enzymes

Telomere

DNA repeats found at the ends of chromosomes

1.Prevents fusion of chromosomes by double strand break repair

2. Replication at the ends of linear chromosomes (Lagging strand utilizes multiple primers, primers must be removed for DNA pol to fill in gaps, however Primer found at end of lagging strand unable to fill in gap since 3’ → 5’, meaning during Replication will always lose genetic material on lagging strand, this is solved by adding Telomere at end of Chromosome

What is the function of enzyme Telomerase

When ends of Telomere have been “eaten away” Telomerase is able to make copies to prevent loss of genetic material

Telomerase (reverse transcriptase) recruits telomerase RNA, Telomerase will use telomerase RNA as template to add complementary nucleotides

Distinction with Telomerase is that it’s not autonomous, instead two different genes encode for telomerase RNA and telomerase

Transcription

DNA-dependent RNA polymerization '“meaning using DNA as template to make RNA”

Distinctions between DNA and RNA polymerization

Promoter is apart of the gene (acts as binding site for transcription of only genes, not the entire genome), not apart of mRNA or RNA being transcribed from gene tho

What is the function of a promoter in transcription?

Promoter (piece of DNA seq, apart of gene) determines direction where to start transcription, which DNA strand is template, how many copies of RNA made

How is the Promoter region recognized?

Recognized by a transcription factor, binds to template strand. RNA polymerase recruited/binds to transcription factor, RNA polymerase initial nucleotide marks the Transcription start site (TSS) with +1 (numerical values assigned both sides)

Promoter determines orientation/direction of transcription

Consensus seq in E.Coli Promoters

Mutation in (-35box) and (-10 box) results in

Functional elements in a promoter

1000 bp upstream of the transcription start site recognized as the Promoter, however within Promoter find functional elements (short DNA seq recognized by transcription factors)

Types of Functional elements

Common consensus (Ecoli -10 and -35, Eukaryotes TATA box)

Gene specific elements

CDNA Library, How to determine which genomic regions are protein-coding genes?

Extract many RNAs, will take all mRNA into single test tube (every single mRNA has 5’ cap and 3’ end has poly A tail)

Add RNA oligos (short piece of RNA single strand) to 5’ end with Ligase

Add poly-T primer (DNA primer) and Reverse Transcriptase (use RNA as template to make DNA)

Add RNase to digest RNA and left with single stranded DNA called cDNA (complementary to RNA)

Design primer thats complimentary to cDNA and DNA pol

Single stranded DNA into double stranded DNA (ds cDNA)

All ds cDNA corresponding to RNA from cell

Undergo PCR (Denature, Anneal, Amplification), since know both ends, can introduce restriction sites to two ends, digestion, and ligation, introducing cDNA fragment into a vector

Transform vectors into E.coli, transform into plate, forming many colonies

Sequence each colony, getting cDNA seq matches mRNA seq

Able to determine Transcription start site

For a specific gene, we know its cDNA/RNA seq.

Want to know how much RNA produced (transcriptional activity of a promoter/ promoter strength) in different cell types under different conditions (more RNA produced more proteins produced)?

1. Northern blot

2. Transcriptional Reporter Assay

Northern blot experiment

Detect a specific RNA and its relative level

Given known mRNA sequence, make RNA or DNA probe (complementary sequence to small region of specific RNA, that is radioactively labeled at 5’ end)

Extract many RNA, undergo Gel electrophoresis (separated based on size)

RNA found inside the gel, however want to transfer to surface, do so by using Nylon membrane, incubate in solution of probe

Probe binds to RNA (hybridization), able to detect RNA due to radioactive label to determine size of mRNA (based on position), relative mRNA level (intensity of band)

Transcriptional Reporter Assay

PCR promoter region ONLY, clone into plasmid, immediately upstream of the coding region of a reporter gene, if promoter activated in cell (transcription factor bound and recruits Poly), coding region will be transcribed into report mRNA, reporter mRNA will be transcribed into reporter protein (easy to detect) able to determine transcriptional activity

How to identify functional elements (short DNA pieces in promoter that determine transcriptional activity)?

Deletion series or Linker Scan

Deletion series

Take promoter, using report assay clone into plasmid or Northern blot, look at activity of promoter (should be 100%), make promoter shorter (if transcriptional activity still 100%, that specific region of promoter not important, make shorter and if activity decreased to 50% determine this region is a functional unit, and continue…until 0%)

Linker Scan

Clone out promoter into a reporter, instead of removing part of promoter, mutate reporter portion of reporter and continue…

Difference in experimental results between Deletion series and Linker Scan

With deletion series always compare to fragment before, not the original full length reporter. Whereas with Linker scan each mutant compare to wild type or original promoter

Goal To identify transcription factors that bind to a specific promoter?

EMSA (Electrophoretic mobility shift assay)

DNase footprinting

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EMSA (Electrophoretic mobility shift assay)

Know before experiment is gene of interest

PCR out promoter region, introduce radioactive p32 label

Fractionate proteins from cell lysate (separate proteins), put into test tube

Mix radioactive gene with test tube

Run Gel electrophoresis (this gel is non-denaturing or native gel), will notice one band at the bottom which corresponds to promoter (not bound to protein migrates fast), if you notice two bands (one corresponds to promoter bound to protein and a lighter band being just promoter)

DNase footprinting

Before experiment know promoter region and transcription factor. However, want to identify specific binding site of transcription factor

Radioactive label one end of sDNA strand, achieved by designing primer (one has label, other does not)

Get shiz ton of copies, split into two test tubes

One test tube mix with transcription factor, every single copy of DNA will be bound with TS

Other test tube do not add TS

Will add little DNAse (this specific DNAse cuts at multiple random sites of DNA) treatment to both test tubes. Find that in test tube with TS the DNAse cannot cut where TS is bound

Run Gel electrophoresis (denaturing gel, removes protein and unlabeled DNA fragment)

How to determine exactly where the transcription factor binds? Will need to be told labeled DNA end (# away from TSS) as well as DNA fragment size marker(means distance from labeled end)

How do we know whether DNA binding proteins activate transcription?

Some proteins activate, inhibit, or have no effect, thus have to test through In vitro Run-off transcription assay

In Vitro Run-off transcription assay

What’s needed in a test tube?

dsDNA (template) gene which contains promoter

NTPS (p32 labeled)

RNA pol

Two test tubes one with (+) and one without (-) transcription factor of interest

Prokaryotic transcription

only 1 RNA pol (makes all RNAs)

Eukaryotic transcription polymerase

Which RNA pol contains CTD and what is the function?

ONLY RNA pol II contains a large subunit C-terminal domain end (CTD), CTD phosphorylation is critical for transcription initiation, Phospho-CTD is critical for co-transcriptional mRNA processing

What is the function of RNA pol I?

Responsible for pre r-RNA (ribosomal RNA, consists of 18s, 5.8s, 28s) → major components of ribosomes (however, is not an information carrier instead functions more like enzymes)

Find r-RNA cluster gene repeats throughout specific regions of chromosomes

Structure of rRNA gene (this is DNA, not RNA since has not been transcribed)

Consists of promoter region, transcription start site, coding region

Promoter region (CORE element (binds to transcription factor called CF) and Upstream element (binds to transcription factor called UAF) both transcription factors linked by TBP (TATA box binding protein)

What happens when TBP is linked to CF and UAF transcription factors?

Will activate transcription, will transcribe coding region called pre-rRNA (pre since not processed yet), Will find snoRNA/small nucleolar RNA (form bp to specific region of pre-rRNA)

What is the purpose of snoRNA?

Recruits enzymes to modify pre-rRNA by making nucleotide modifications (add chemical marks), if do not have these modifications wont be able to assemble ribosomes

What is the role of RNase?

RNase cuts pre-rRNA into different fragments, will continue to do so until isolate 18s, 5.8s, and 28s segments

What happens to the 18s pre-rRNA?

Will be associated/bind with ribosomal proteins forming small subunits of ribosome

What happens to the 5.8s and 28s pre-rRNA?

Will form bp with one another and associated with 5sRNA (only RNA made with Pol III) and ribosomal proteins forming large subunit of ribosome

Important to know that modifications of rRNA occur co-transcriptionally in the nucleolus

co-transcriptionally (transcription of pre-rRNA DNA is accompanied by modifications as well)

All these steps occur in Nucleolus (region of Nucleus)

What is the structure of RNA Pol III

Makes tRNAs, 5s rRNA (important distinction between these two structures is that promoter is encoded in the RNA being transcribed since is downstream of transcription start site

What is the structure and function of RNA pol II?

Pol II makes all the mRNA,

TFIID binds the TATA box promoter and helps recruit RNA polymerase II.

TFIIH then unwinds the DNA and phosphorylates the Pol II CTD domain, which activates RNA Pol II and allows transcription to begin.

They are called general transcription factors (GTFs) because they are required for transcription of most Pol II genes.

(TFIID (consists sof TATA box binding protein + TAFs) and TFIIH (consists of DNA helicase and Pol II CTD kinase protein))

mRNA processing

5’-capping and after 3’-polyadenylation

5’ capping

Once the RNA polymerase II CTD is phosphorylated, it recruits:

• Capping enzyme (CE) → adds a GTP cap to the 5′ end of the pre-mRNA

• Methyltransferase (MT) → adds methyl groups to form the 5′ methyl cap

• Function is to mark mRNA for translation by ribosomes

3’polyadenylation

5’ has undergone 5’-capping, find that Specificity factor (SF) binds to AAUAAA sequence and Cleavage stimulatory factor (CSTF) binds to G/U 3’ end.

Pol-II CTD phosporylation once again allows for 3’-polyadenylation, however requires AAUAAA sequence and G/U as well to form poly A tail

What happens when both SF and CSTF are bound to specific cite?

Another protein will bind in between both called Cleavage factor (CF), which causes a cut in between. Find that CSTF is gone, only SF remains attached.

What happens after CF has made a cut in between?

SF recruits another protein at 3’ end called poly A polymerase (PAP), which is activated by SF and add many A nucleotides to 3’ end

What happens after the PAP has added many A nucleotides to the 3’ end?

Another factor called Poly-A binding protein II (PABPII) will further activate PAP to add a total of 300 A nucleotides

What is the function of the addition of A nucleotides at 3’ end?

1. Marks mRNA for nuclear export and translation

2. Protects mRNA from degradation

Only Pol II transcribed genes can have 5’ capping since Pol-II CTD domain recruits enzymes, all other RNA such as r-RNA, t-RNA does not have 5’ capping

How come snRNA (small nuclear RNA) are transcribed by Pol-II, however only contain 5’ capping, but not poly A tail?

If has Pol II promoter, will be transcribed by Pol II which is ONLY needed for 5’ capping, however snRNA is missing AAUAAA and G/U sequence thus will have no poly A tail

Recap of mRNA processing through Pol II Promoter

Say you PCR rRNA gene and put it under Pol II promoter, which Pol will be used to transcribe gene?

Will use Pol II since contains Pol II promoter gene sequence thus will have 5’-capping also but not 3’-poly-adenylation

mRNA splicing

Note that mature mRNA (doesnt have introns)

5’UTR and 3’UTRare transcribed and kept in mRNA however are not translated

What is a UTR? Are UTR found in the introns or exons? Are UTR translated?

UTR is an untranslated region, they are found in exons, and they cannot be translated

Experiments to understand splicing of mRNA

1. Take single DNA template strand and mix in test tube with mature mRNAs that are transcribed by this DNA and look at EM. Able to understand direction based on poly A tail

In Vitro splicing analysis to determine biochemical steps of splicing (substrate/precursor (pre-mRNA) → intermediate(?)→ product (mature mRNA)). Goal to determine intemediate

DNA template/ P32 labeled pre mRNA generated by in vitro transcription, pre-mRNA transcribed from DNA (don’t get mature mRNA since this occurs in the test tube not in a cell, dont have the machinery), pre-mRNA incubated with nuclear extract (enzymes needed for splicing), perform reaction under different time points, and run through Gel electrophoresis (mobility dependent on size of fragment but also structure of molecule)

Biochemistry of In-Vitro splicing

What role does spliceosome play in Intron recognition?

Spliceosome (composed of snRNAs (majority produced by Pol II besides U6 snRNA produced by Pol III) + proteins) → Form ribonucleoprotein (RNPs)

What recognizes and removes the intron?

U1 and U2 snRNAs of Spliceosome

What role does U1 play in intron recognition

Will recognize consensus sequence found in boundary between exon and intron, form complementary sequence

What role does U2 play in intron recognition

Will recognize and form bp around the branch point (but avoid the A)), U2 snRNA will bind to U2AF which required to recognize ending point of the intron

Splicing will occur once U1 and U2 snRNA identify introns and find …

5’ splice site at U1 snRNA and 3’ splice site at U2 snRNA

Steps of splicing pre-mRNA

RNA helicase (utilizes ATP, to break H bonds allowing for conformational change of spliceosome subunits) → assembly/disassembly/remodeling of spliceosome subunits → splicing

Once 5’ splice site and branch point/3’ splice site recognized, more snRNPs and will bind to these sites to form Spliceosome

Will undergo conformation change allowing branch point (A nucleotide 2’ OH group attacks phosphate sugar bond between exon and intron triggering first transesterificatrion)

Will undergo further conformational change, regenerating introns 3’ hydroxy group, this OH group will attack phosphate sugar bond between end of intron and beginning of Exon 2, triggering second transesterifcation. Cutting intron

Splicing occurs co-transcriptionally

As Pol II is transcribing mRNA, splicing is occurring as well.

Pol II CTD domain recruits Spliceosome and cuts off intron

Pol II-CTD → spliceosome (must recognize 5’splice site/3’ splice site/branch point) → Splicing

Which step in the splicing reaction would be inhibited if you generate a pre-mRNA containing a 2’ deoxy adenosine (dA) at the branch point A?

First transesterification

Functions of some Pol-III transcripts

5S rRNA → 60s subunit (large) role in translation

tRNA undergoes aminoacylated → tRNA-aa plays role in translation

U6 snRNA → Spliceosome (with snRNAs generated by Pol II) plays a role in pre-mRNA splicing

For both tRNA gene and 5s-rRNA gene the promoter is downstream of TSS, meaning transcribed during translation

Not all Pol-III downstream

What processes does pre-tRNA undergo?

5’ cleavage triggered by RNase P (Ribozyme:RNAs with enzymatic activity), splicing of introns, 3’ trimming, and base modifications

Not all ribonucleoprotein complex (RNPs) are ribozymes, which are tho…?

Spliceosomes and Ribosomes

Nuclear export

Pol I transcribed rRNA and ribosomal subunit synthesis both occurring in Nucleolus, after Ribosomes are formed are exported out of the Nucleus because function is translation and that occurs in cytoplasm

Pol II synthesize mRNA, undergoes 5’ capping, splicing, 3’ polyadenylation all occurs in Nucleus and exported out nucleus (mRNA triggers translation in cytosol and meet with ribosomes). Pol II also makes snRNAs (subunions of spliceosome, but not exported out of nucleus since splicing occurs in Nucleus. Pol II makes snoRNA which will stay in the Nucleolus and make modifications

Pol III make tRNA also apart of translation machinery thus exported to Cytosol

Nuclear export of mRNA

mRNA in the nucleus is bound to many proteins which stabilize it and export it to Cytoplasm, these proteins go back to the Nucleus and once mRNA in the cytoplasm different proteins bound to stabilize and facilitate recognition of mRNA for translation

Steps to determine amino acid sequence of protein translated

1. Start 5’ end at look for AUG(start codon)

2. Everything after considered triplet codon

3. Once reach UAG or UGA have reached stop codon

Genetic code

Universal across all living organisms

Reading frames

The way you analyze the RNA sequence into triplets

In vitro translation

In test tube one synthetic RNA that does not have AUG, may be three possible reading frames (only possible since add abundant concentration allowing for energetically unfavorable reaction to occur)

However, under physiological conc of RNA + amino acids does not work

1 mRNA only have one reading frame (ORF) thus one polypeptide, this is energetically favorable due to 5’cap + AUG (start codon) + 3’ poly A tail

How does the cell match AA to codons?

tRNA molecule (one side nucleic acid with complementary sequence (anti-codon) and other side connect with amino acid)

tRNA transcribed by Pol III

What connects tRNA with amino acid?

Enzyme called Aminoacyl tRNA synthetase (one pocket match with amino acid and other pocket with tRNA)

Amino acid is connected at 3’ end of tRNA

Interesting thing about tRNA anti-codon is that third bp doesn’t need to be complementary (wobble position allows G-U, I-C, I-A, I-U) (I is modified A)

There are 30-50 anti-codons tRNAs, for amino acids have 61 (64 codons in total, but 3 are stop codons)

1 tRNA synthetase → multiple different tRNAs

1 anticodon → different codons

1 codon → different anti-codonds

However, one anticodon can only match with one amino acid

Translation machinery structure

GTP is the energy source

Stepwise synthesis of translation

Initiation, Elongation, Termination

Initiation (pre-Initiation complex)

Translation Initiation process

Pre-initiation complex (small ribosomal unit) recruited by 5’ cap and poly A tail to mRNA

Starts at 5’cap and scan through mRNA and once meet start codon will stop there and recruit large subunit ribosome for Elongation

Translation elongation process

initiator tRNA found in P pocket, when P pocket matches with AUG (Met, start codon) and than will recruit next tRNA through elongation factor into pocket A, once tRNA in pocket A large subunit will catalyze reaction so first amino acid release from first tRNA and will be connected to second amino acid (thus connected to second tRNA), ribosome will move towards the right codon, first tRNA will enter E (exit pocket) and second tRNA moves to P pocket, and repeat

Translation termination process

A pocket matches with one of three stop codon, there will be a protein similar to tRNA that will enter A pocket called release factor 1 and 3 (why stop codon is not translated and instead ribosomal unit dissociates)