Processes to learn for bio Flashcards
Transcription
- Occurring in the nucleus(in Eukaryotes) in cytoplasm (in bacteria), DNA helicase unzips DNA, and RNA Polymerase binds to the promoter region initiating transcription
- RNA Polymerase runs along template strand in a 3’-5’ direction building a complementary template strand in a 5’-3’ direction, stopping once its hits a stop codon(UAG,UGA,UAA) falling off, leaving us with Pre-mRNA.
3.Methyl-g cap is added to the 5’ prime end of Pre-mRNA molecule and a poly-a-tail is added to the 3’ prime end - Spliceosomes cut out introns and splice together exons leaving us with m-RNA.
Alternative splicing
Alternative splicing enables the same gene to produce different proteins.Splicing involves different exon splices where different exons are combined to form several kinds of mRNA, each with a different base sequence.
Translation
Initiation
1. The 5’ end of mRNA molecule binds to Ribosome and is read until the start codon (AUG) is recognised.Then a tRNA molecule with a complementary anti codon (UAC) binds to ribosome delivering the amino acid methionine,signifying the start of translation
Elongation-
2. After the first amino acid attaches, mRNA molecule is fed through the ribosome so the next codon can be matched to its complementary tRNA anti-codon.Complementary tRNA molecules deliver specfic amino acids to the ribosome, which bind to adjacent amino acids with a peptide bond via a condensation reaction.The first tRNA molecule then leaves the ribosome and continues to pick up amino acids, growing the amino acid chain
Termination-
3.Linking of amino acids in the polypeptide chain continues until the ribosome raches a stop codon.The stop codon signals the end of translation.The poplypeptide chain is then released by the ribosome into the cytosol or endoplasmic recticulum.
Trp operon regulation when levels are high
1.To regulate the expression of Tryptophan structural genes, the regulatory gene for trp operon is constantly expressed, producing a repressor protein
2.when levels of tryptophan are high meaning they are present. Tryptophan is able to bind with the repressor protein inducing a confromational change (physical change) in the repressor, allowing it to bind to the operator region.
3.This allows the repressor protein to bind to the operator region
4.Acting as a block, the repressor protein inhibits the transcription of the tryptophan structural genes by RNA polymerase, inhibiting the unnecessary production of tryptophan.
Trp operon regulation when levels are low
1.When Trypophan levels are low, there is not enough tryptophan molecules present that are able to consistently bind to the repressor protein,causing it too detach from the operator region.
3.This allows RNA polymerase to transcribe the trp structural genes so that level of tryptophan can increase
4.However, as tryptophan accumaltes in the cell it will once again bind the repressor protein slowly stopping transcritption of the trp structural genes
5.Together, these mechanisms keep the amount of tryptophan available at a relatively constant level to ensure that energy and resources are expended appropriately.
Trp operon by attentuation when levels are high
- Ribosome runs past the Tryptophan codons present in region 1 of the gene (since tryptophan is present)however, pauses between region 1 and 2 due to a stop codon
2.This prevents region 1 from binding with 2 and forces region 3 to bind with 4, forming a “hairpin loop”
3.This puts tension on the weak attenuator region (Due to adenine and thymine 2 weak hydrogen bonds), and mrna strand is forced too pull away ending transcription
Trp operon by attenuation when levels are low
1.Ribosome pauses at the 2 tryptophan codons in region 1 waiting for a tRNA molecule to bring tryptophan amino acid.
2.This allows 2 to pair with 3 preventing 3 and 4 from pairing with each other
3.while this creates a hairpin loop,its too far away from the attenuator region and therefore mrna doesn’t pull away and the ribosome continues transcribing the genes, creating more tryptophan
How are proteins secreted (full process)
1.Proteins intended for secretion are synthesised at the ribosomes found on the Rough Endoplasmic reticulum.
2.They are then folded at the Rough ER and are transported to the golgi aparatus, via transport vesiciles.
3.At the Golgi apparatus, the proteins are modified before being packaged into secretory vesicles.
4.These secretory vesicles fuse with the plasma membrane, releasing the proteins into the extracellular environment via the process of exocytosis.
Process of exocytosis
1.A vesicle containing secretory products is transported to the plasma membrane via secretory vesicles
2.The membrane of vesicle fuses with the plasma membrane
3.The secretory products are released from the cell into the extracellular environment
CRISPR-Cas9
Exposure:
Bacteriophague injects its viral DNA into bacterium,with cas 1 and cas 2 (endonuclease, enzyme that cuts DNA)cutting out the PAM sequence(protospacer region) of viral DNA.
Expression:
This Pam region then gets inserted between the repeater regions of the CRISPR sequence and is transcribed,becoming guide RNA
Extermination:
GRNA attaches to cas9 (endonuclease) which forms a Crisper-cas 9 complex.This is Guided to attack any renterring viral DNA since it has a complementary sequence.The next time viral DNA enters the bacterium, The CRISPR cas-9 complex identifies the viral DNA and the cas 9 cuts sugar phosphate backbone of the viral dna, inactivating the virus.
CRISPR-cas 9 in gene technology
1.Synthetic gRNA is made in a lab containing the complementary sequences to the target gene that scientists wish to cut ,Cas9 an endonuclease with an appropriate pam sequence is then identified
2.Synthetic gRNA is then mixed with cas-9 to form the CRISPR-CAS9 complex,this formation is then injected into a specific cell such as a zygote
3.The Cas9 finds target pam sequence and using the grna, checks to make sure it has complementary sequence
4.Cas 9 works and cuts at the restriction sites leaving blunt ends,Scientists can then introduce new nucleotides hoping it will ligate back into the DNA
5.DNA attempts to ligate back together
PCR
Denaturation:
DNA is heated to approximately 90-95c causing it to denature,breaking the hydrogen bonds between the bases to separate the strands,forming a single stranded DNA.
Annealing:
The single stranded DNA is cooled to approximently 50-55c to allow the primers to bind to complementary sequences on the single-stranded DNA
Elongation:
The DNA is heated up again back to 72c which allows taq polymerase to work optimally. Taq polymerase binds to the primer which acts as a starting point and begins synthesizing a new complementary strand of DNA
Repeating:
steps 1-3 are repeated multiple times to create more copies of DNA
Gel Electrophoresis
1.Dna is cut by restriction endonucleases or a short sequence of DNA that has been amplifieid during PCR
2.DNA samples are placed in wells within an aragose gel (Negative side),The aragose gel has tiny pores that allow gel to pass.The aragose gel has been immersed in ion rich buffer allowing an electrical current to pass through, a standard ladder is also added to determine length.
3.An electrical current one positive and one negative is passed through Gel.Since DNA has a negative charge (due to its phosphate backbone), it moves towards the positive end.
4.Smaller DNA fragments move faster through the Gel and travel further than larger fragments, after a few hours current is switched off , as DNA fragments stop moving they settle into bands
5.DNA is difficult to see so its stained with a florouscent dye such as ethidium bromise which allows bands to be viewed under UV lamp.
Recombinant plasmid (Circular DNA vector that is ligated to incorporate a gene of interest, edited to integrate a target gene)
- Gene of interest is isolated and copied using PCR
-The Gene of interest cannot have introns prior to insertion,since prokaryotes dont have introns and therefore bacteria wouldn’t know what to do with intron segments,Introns are typically excluded from the gene of interest via two different methods; synthetic DNA, which is made in a lab by scientists without introns and cDNA (copy DNA) which is made by a an enzyme called reverse transcription which functions to transcribe mRNA backwards into cDNA
- Plasmid vector is selected into which gene of interest will be inserted
Plasmid vectors contain 4 essential parts
- Antibiotic resistance genes- (e.g ampR,tetA) This antibiotic resisitance that the plasmid vector allows it too be seen even after the antibiotic is added to the recombinant plasmid later on
- Origin of replication (ORI)-Sequence that signals the start site for DNA replication in bacteria
- Reporter gene- Genes that have an easily indetifiable phenotype, that a
- DNA of plasmid is cut at one point by restriction endonucleases, that create sticky ends, This changes the form from circular to linear.
- The gene of interest is then cut using the same restriction endonucleases that cut the plasmid, too ensure that the plasmid and gene of interest have complementary sticky ends
- The gene of interest (Foreign DNA fragments) and plasmids are then mixed and in some cases, their sticky ends pair by weak hydrogen bonds, the complex is now a recombinant plasmid
- The plasmid may not accept the pairing and therefore just reseal without the foreign DNA transforming back to just a plasmid, however if the Plasmid accepts,Ligase is added and this makes the joining permanent through covalent bonding.
antibiotics can be used to identify whether a plasmid has taken up the gene of interest
How to encourage plasmid to accept inserted gene
Heat shock:Using temperature to increase permiability of plasmid membrane:
1. Bacterial culture is placed into ice bath ,Recombinant plasmids with the antibacterial resistant allele (i.e tetR) are added to bacteria culture which is chilled
2. The bacteria and plasmid is then heat shocked by being placed into hot water around 42c for 50 sec, this heat shock causes the plasma membranes bacterial cells to alter, increasing the chance of the cell uptaking the plasmid
3. The mix is then returned to ice bath to return plasma membrane to natural state
4. The bacteria is then plated on a agar plate along with antibiotic (i.e tetracyline) this is then incubated at 38c overnight.
5. Bacteria that have no taken up the plasmids will be killed by the antibiotics but bacteria that have taken it up will not since they inhabit the antibiotic resistant alleles from the plasmids.
6. Bacterial cells that have taken up the plasmids will be selected and transformed, replicating
or
Electroporation:
1.Electrical current is passed through solution containing plasmid and bacteria,Electrical current causes the plasma membrane to become more permeable allowing plasmid vectors to cross through the plasma membrane
