Molecular Cell Biology Midterm 1

Process of PCR

Process of PCR

Amplification of a gene

• heat dna, taq, primers, and dNTPs To denature the DNA

• Cool to anneal primers

• Heat again to optimal temperature for taq polymerase

An entire genome can be represented in a DNA _______

library

What other genetic material can you use for PCR

MRNA to cDNA

• Add first primer, reverse trasncriptase and dNTPs

• Allow complementary DNA strand to be made

• Separate strands and add second primer

• Do PCR to create cDNA copies

Peptide bonds can _______ about the peptide bonds

Rotate

What is the primary structure of a protein

Aa sequence

what is the secondary structure of a protein

Local 3D structure (beta sheet, alpha helix)

What causes secondary structure

H bonds between distant amino acids

What is the quaternary structure of a protein

3D structure of a protein (globular)

• made up of multiple peptides

In the alpha helix, every ___ (#) amino acid shares a hydrogen bond

4th

In an alpha helix, the side chains are oriented _______

outward (no interference)

In the beta sheet, there are 2 or more stretches of ________ amino acid sequences

Parallel

Hydrogen bonds between ________ amino acids (they can be more than 4 aa’s away)

adjacent

In the beta sheet, the side chains are oriented ______

outwards

Proteins are classified into _______

Families (related proteins)

what is similar between proteins of the same family

Domains

_________ structure is very similar among proteins of the same family

Tertiary

What is a protein domain

Give a protein a particular function

T or F: a protein can have multiple domains

T

More domains, means increased _______

Complexity

Function of protein kinases

Add phosphate group

Function of protein phosphotases

Remove a phosphate group

What 3 amino acids are most affected by phosphorylation

serine, threonine and tyrosine

What is a GTPase or GTP-binding protein

Bind and hydrolyse GTP

• GTP-bound: active

• GDP-bound: inactive

What 2 proteins control the activity of GTPases or GTP-binding proteins

GAP (GTPase activating proteins) and GEF (Guanine exchange factors)

Function of GAP

Hydrolyzes GTP, forming GDP

• promoted GDP bound state (inactive)

Function of GEP

Exchange of GDP to GTP on GTPase

• activating regulatory protein

What was the old way of purifiying proteins versus the new way

old: fractionation and combine it with in vitro assays (kornberg)

New: immunopreciptiation

Process of immunoprecipitation

1. Grind up cells to maintain extract

2. Add an antibody against POI

3. Add agarose beads coupled to the antibody

4. Antibody bead complex binds the POI

5. Centrifuge and wash, protein will be in supernatant at bottom of tube

What type of purification is used if there is no antibody for POI

1. Clone gene and express it in ecoli

2. The plasmid vector will have a strong inducible promoter

3. Epitope or affinity tag sequence can be incorporated to purify DNA

Process of SDS page

1. Proteins are heated with detergent is used to charge the DNA molecules and get rid of their tertiary structure

2. Loaded onto gel and separated based on size

3. Dyes can be used to detect protein quantity (darker = more protein present)

A specific known protein can be detected using a _______________

Western blot (need antibody against protein)

A specific unknown protein can be identified using __________

mass spectrometry (cut out piece of gel with lots of protein

How do we purify proteins expressed in E.Coli

1. Clone the gene into a vector containing an affinity tag or epitope tag to form a fusion protein

2. Express it in bacteria or animal cells

3. Purify it with affinity chromatography

what is an epitope tag

Small protein sequence that can be recognized by commercially available antibodies (example: HA tag)

• sequence can be incorporated into primer

What is an affinity tag

longer sequence that must already be present in the plasmid

• can bind to a specific substrate allowing for purification by affinity chromatography

How to make a fusion protein

PCR (incorporate epitope sequence into primer) and clone (use vector plasmid with affinity tag sequence already in it)

process of GST affinity purification

1. Clone gene into the GST vector

2. Express the fusion protein

3. Add agarose beads coupled with glutathione (GSH beads)

4. GST has a high affinity to glutathione and will bind

5. Wash and elute with excess glutathione to complete for binding to GST

6. The fusion protein is now purified

process of GST pull-down

1. Bind protein X to GST and protein Y to HA

2. The GST protein X complex will bind to GSH beads on column

3. Incubate column with HA protein Y complex

4. Wash and elute with GSH

5. If protein Y bonded to protein X on column, it would not be washed away

6. Run on protein gel

What does GST pulldown test for

Interaction between 2 known proteins

Process of co-Ip

Same as immunoprecipitation but a less aggressive buffer is used to preserve protein-protein interaction

• Protein X is bound to GSH bead and eluted

• After wash, protein Y will also be bound to protein X-GSH bead complex

• Can then use western blotting to identify interacting protein (known) or mass spec (unknown)

How are unknown proteins analyzes using mass spec

After co-IP, run the eluded product on SDS gel

Stain the gel with stain

Cut out the most prominent band and perform MS on it (contains protein that was specifically eluted, the lighter band contains the co-IPed protein

Use peptidsase to cut up proteins into a mix true which is ran through MS

MS uses mass to charge ratio to indenting potential amino acid sequences of proteins

Compares that to known proteins to figure out which one it is

What 2 methods are used for testing for interaction between two proteins

Co-IP, western blotting and GST pulldown

what method is used for identification of unknown interacting proteins

Co-IP and MS

What databases are used to identity unknown proteins after MS

1. BLASTP: Database used to identify related proteins in same or different species

2. Domain prediction: identifies potential domains based on sequence similarities to characterized ones

What method is used to study quaternary structure or proteins

X ray crystallography

Process of X-ray crystallography

1. express protein in ecoli and purify it with affinity purification

2. Purified protein solution is dehydrated into crystal

3. X rays are shot at it and their diffraction patterns determine its structure

How are polyclonal antibodies generated

1. Inject protein and inject it into animal

2. Wait a month ad take serum which contains antibodies for POI

3. Serum will contain antibodies for multiple regions of protein

4. Specific proteins can then be purified from mixture using the reverse of IP

5. Incubate serum with beads bound to specific protein that binds antibody

how are monoclonal antibodies generated

1. Inject mouse with proteins

2. Wait a month and then remove spleen and harvest plasma B cells

3. Fuse B cells with myeloma cells (Hybridoma cell)

4. Break cells up and centrifuge them

5. The supernatant will contain pure monoclonal antibodies

How are antibodies I used to detect specific proteins in a cell

1. Immobilize protein onto a surface

2. Incubate protein with the primary antibody (specific to protein)

3. Incubate protein-AB complex with secondary antibody that is coupled with a fluorescent marker (from a different animal)

   • if primary AB is from a rabbit, then the secondary AB must be produced in another animal (goat anti-rabbit AB)

process of live imaging

1. Use GFP tag

2. Clone POI into plasmid already containing GFP sequence

3. Transfect into other animals

4. Image fluorescent cells with light microscope

How is RNAi used for regulation of gene expression

1. Dicer complex cleaves original dsRNA into 21-23 nt fragments called siRNA

2. RISC processes siRNA into guide strand and passenger strand

3. The RISC guide strand complex binds to complementary mRNA and cleaves it

What is shRNA

SsRNA that can fold back on itself and become double stranded

• in viruses and retroptransosons

• RISC can process this and develop a guide RNA which can be used to fight off this infectious agent

explain how RNAi was discovered

• researchers predicted that combining anti-sense and sense RNA would reduce gene knockdown because they would bind each other instead of the host RNA

• Turned out to be the opposite (gene knockdown 100x stronger)

• Instead of simply having the introduced anti sense mRNA bind to the self proteins, the antisense and sense strands bound together creating dsRNA

• This leads to RNAi

Explain structure of CRISPR array

Regularly interspaced palindromic repeats separated by spacer sequences

• spacer sequences correspond to sequences in plasmids and viruses

what are CAs proteins

CRISPR associated proteins

• RNA binding, helicase and DICER ability

• Can recognize and bind incoming viral rna that matches spacer sequence as well as cleave it

Overview of CRISPR process

CRISPR array is transcribed into pre-crRNA

Pre-crRNA is cleaved by CAs proteins to form crRNA

Other CAs proteins bind this RNA

Explain the structure of a type II CRISPR array

• Cas1, Cas2, and Cas9 are involved in processing the viral DNA

• They are located upstream of crispr array along with tracrRNA

Functions of Cas9 and cas1 and 2

• Cas9 associated with crRNA to mediate target recognition and cleavage

  • Recognizes PAM sequence (only found in viral plasmid DNA)

  • Also processes pre-crRNA into crRNA

• Cas1 and 2 will act as endonucleases to cleave the viral DNA at the 5’ ends around 35 nts upstream of the PAM sequence

Function of tracrRNA

• Tracr RNA is a non-coding rna with a region that is complementary to the CRISPR repeat sequence

Structure of crRNA

• One spec are and the adjacent repeat sequence

• Associates with tracrRNA and Cas9 to act in immune system

CrRNA + TracrRNA = ___________

guide RNA

How does the CRISPR DNA protect itself from being recognized by Cas9-guide RNA complex

It does not contain the PAM sequence

How is CRISPR/Cas9 used for gene editing

• Design synthetic guide RNA

  • Identify PAM sequence in GOI and use sequence 20nt immediately upstream of it for complementary DNA

  • Should also contain the CRISPR repeat And tracrRNA

• Introduce cas9 into cells via transfection As well as the guide rna

• The complex will form in cells and cleave the GOI (ds break)

  • if NHEJ is used: there will be a deletion made

  • For HEJ to be used: have to flood the with plasmid containing complementary sequence containing some type of alteration- affinity tag, mutation etc

What was kornberg’s first experimental result

1. Grind up ecoli cells and obtain cell extract

2. Add radioactive thymine + all nts and template dna

3. Waited for DNA synthesis to occur

4. Added percholric acid to stop the process And precipitate the DNA (Nts are not precipitated)

5. However, he found radioactivity int he pellet indicating that there was an enzyme synthesizing the DNA from dNTPs

What was kornberg’s second experimental result

1. Fractionation was used to separate proteins by size and function

2. Test every fraction for dna synthesizing ability And use successive fractionation until somewhat pure

3. Started dna synthesis experiment with cold nts (not radioactive)

4. Added short pulse of radioactive thymidine (last nt added would be radioactive)

5. Added 3’→5’ exonuclease

6. Added percholric acid to precipitate DNA

7. Radioactivity was present in the supernatant meaning that the radioactive dNTP was cleaved

8. This indicated that nucleotides were added 5’→ 3’

9. When a 5’ → 3’ exonuclease was used, the radioactivity remained in the pellet

What was kornberg’s third experimental result

1. Added primers , 4 dNTPs (had a radioactively labelled C in the base of the nt)

2. Also radioactively labelled the terminal phosphate

3. After DNA synthesis he saw that the radioactivity of the phosphate moved from the pellet to the supernatant, and the base remained in the pellet

4. The radioactivity of the phosphate was equal to that of the 2 labeled carbons, indicating that 2/3 Pi were removed

5. Indicated that a pyrophosphate was lost during addition of nts in new strand

The DNA replication fork is ____________

asymmetrical (continuous in 3’→ 5’, discontinuous in 5’ → 3’)

okazaki’s experiment for lagging strand

1. Extracted ecoli dna and added short pulse of radiactive thymidine to label DNA

2. then added alkali to stop the process

3. Used a sucrose gradient to separate DNA by size

4. Measured radioactivity in each fraction

5. 30s: Graph showed two peaks, one showing large and one showing small fragments- indicated that the lagging strand was synthesized by connecting small fragments

6. 60s: larger fragments who more radioactivity that shorter (this is because the Okazaki fragments have time to be ligated)

Okazaki and DNA ligase experiment

1. Pre existing temperature sensitive mutants for ligase gene

2. To mutant ecoli cells and allowed thm to replicate dna under permissive and restricted temps

3. Restrictive group: high radioactivity occurs in small fragments because ligase could not connect the Okazaki fragments

4. Permissive temp: far less radioactivity in small fragments because they were being joined by ligase

explain synthesis of lagging strand

1. Rna primase places primer which DNA polymerase extends in the 5’ → 3’ direction

2. Another dna polymerase removed primer and fills fap

3. Dna ligase joined backbone together

okazaki’s experiment for RNA primer

1. Added RNAse which degrades primers during reaction

2. Result: synthesis of lagging strand did not occur (high radioactivity in big fragments)

1. Also added radioactive uracil in separate experiment

2. Found that radioactivity was only present in lagging strand (only small fragments)

Major component of dna replicaiton

Helicase: opens helix

SS binding proteins: binds to single stranded dna and protects it from degradation

Sliding clamp: makes sure DNA polymerase does not fall off strand

Clamp loader: loads sliding clamp onto molecule using ATP

DNA polymerase

DNA ligase

3 mechanism to ensure accurate DNA replication

1. Base pairing before attachment (ensure correct base is added)

2. Exonuclease activity

3. Mismatch repair (if exonuclease activity fails)

explain process of mismatch repair

MLH1 and MSH2 recognize mismatched bases and make a single stranded nick to remove damaged dna

Dna polymerase and ligase activity

The ______ _________ _________ recruits other proteins to the replication origin during G1

Origin recognition complex (ORC)

Explain how telomerase works

1. Due to removal of last rna primer on lagging strand, there is an overhang from the leading strand

2. This ss overhang is stabilized by Sheltrin which recruits telomerase to the site

3. Telomerase has a built in rna primer

4. It uses this to extend the leading strand with many repeats

5. Dna polymerase can then extend the lagging strand

How were RAD mutants used to discover dna repair mechanisms

goal was to identify genes necessary for DNA repair

1. Start with plate of yeast colonies that are highly mutated at random places throughout the genome

2. Stamp onto replica plates, one is control, other is grown under UV light to identity RAD mutants (sensitive to UV light)

3. These mutants have mutations in dna repair genes and will not survive

4. Can trace back the colony that is not present on the RAD plate but prevent on control and study where the mutation took place and in what genes

2 general categories of DNA damage

Chemical alteration of nt (Deamination, depurination)

DsDNA break or ssDNA break

UV light increases ___________, nitrous acid increases __________ and X-rays increase _________

pyrimidine dimers, cytosine deamination, dsDNA breaks

What is depurination

Deletion of purine base (if not repaired can cause a nt deletion in one strand)

What is deamination

loss of amino group from base (if not repaired it can cause a C→ U transformation)

• results in one strand of DNA having a U-A base pair

Pyrimidine dimers

Two adjacent pyrimidines covalently bond, causing bump in dna strand

• dna and rna polymerase cannot pass

3 methods that detect an altered or missing base

1. BER

2. NER (general and TCR pathway)

3. Translesion repair (error prone)

2 methods that detect and repair double stranded breaks

HEJ (error free)

NHEJ (error prone)

BER mechanism

1. Glycolase specific to the type of altered base will detect and remove the altered base

2. AP endonuclease and phosphodiesterase remove the sugar phosphate backbone after base has been removed

3. Dna polymerase and ligase fix the rest

4. Repairs depurination and deamination

NER mechanism

1. XPC-Radner scans DNA and recognizes the dimer

2. Recruits XPG to site which excises the dimer

3. Dna poly and ligase fix the rest

When people have mutation in proteins involved in NER pathway, they develop ________

zerdermapigmentosa

Translesion repair pathway

1. Sliding clamp and DNA poly complex encounter dimer which causes a conformational change

2. DNA polymerase falls off and is relocated by translesion polymerase that excises dimer and inserts ‘best guess nts”

3. Error prone pathway that induces mutations

When is the translesion pathway used

If the NER pathway is not functional (mutation in XPC, XPG or RAD23

TCR pathway

RNA polymerase encounters dimer and stalls

CSB protein recognizes stalled RNA poly and recruits XPG

XPG excised damage

DNA poly and ligase fix the rest

What is cockayne syndrome

Not as severe as XP

Less effective NER

Why is it that there are more mutations in introns than exons

TCR pathway- works to fix damage while genes are being transcribed

Individuals with xeroderma pigmentosa have a present/absent general NER pathway and a present/absent TCR pathway

absent, absent

Individuals with cockayne syndrome have a present/absent general NER pathway and a present/absent TCR pathway

Present, absent

NHEJ mechanism

1. Ku proteins process ends slightly before ligation leading to small deletion

HEJ mechanism

1. Mre11 nuclease complex is recuited to ends of DNA in break

2. Recruits BRCA1 which digests the 5’ ends of broken DNA, creating 3’ overhangs

3. Rad51 mediated homolgous strand invasion

4. Dna polymerase extends 3’ end

5. Dna synthesis of other strand is completed

6. Dna ligase joins ends together

2 important discoveries that led to discovery of CRISPR

1. Regularly interspaced repeats

2. When an infection occurred, a piece of the viral dna was cleaved and incorporated in between repeated

Explain the sequence of events used to identify an unknown interacting protein of a known protein

• Use PCR to amplify the gene (primers contain restriction sites and epitope tag)

• Insert gene using restriction enzymes into plasmid

• Transfect the cell and allow it to express the gene

• Crush up cells and purify the proteins using immunoprecipitation

  • Interacting proteins will bind to the protein attached to the antibody-agarose bead complex

• Use SDS page and a western blot to test whether or not the known protein is bound to the original protein

Explain the sequence of events used to identify an unknown interacting protein of a known protein

• Use PCR to amplify the gene (primers contain restriction sites)

• Insert gene using restriction enzymes into plasmid containing a GST sequence

• Transfect the cell and allow it to express the gene

• Crush up cells and purify the proteins using GST pulldown

  • Interacting proteins will be bound to initial protein after washing

• Use mass spec to identify the sequence of the interacting protein