Molecular Biology Dna And Rna

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Semi-conservative replication

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DNA synthesis uses each strand of the parent to make a new strand of DNA.

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Number of hydrogen bonds in C:G base pairs

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3

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Number of hydrogen bonds in A:T base pairs

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2

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What is required for DNA synthesis in vivo?

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1. Template
2. Primer with 3'OH
3. Building blocks of 5' dNTPs
4. Energy in phosphate bonds
5. DNA polymerase

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What additionally is required for DNA synthesis in vitro?

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1. Mg
2. Buffer
3. Taq DNA polymerase

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What is the proofreading mechanism found in DNA polymerase?

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3'-5' exonuclease activity

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Taq polymerase

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1. Heat stable
2. Non-proof reading
3. Used in in vitro PCR reactions

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Reverse Transcriptase

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Makes DNA from an RNA template and inserts it into genome.

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Telomerase

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Reverse transcriptase found in human stem cells and cancer cells. it prevents telomere shortening using its own RNA template to extend DNA.

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Topoisomerase I

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Relieves strain due to supercoiling by cleaving one strand of the DNA and releasing pressure.

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Topoisomerase II

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Relieves strain due to supercoiling by cleaving both strands of the DNA and releasing pressure.

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Gyrase

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Topoisomerase II found in bacteria that is an isoenzyme so can be targeted specifically by flouroquinone that will not affect the human.

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Euchromatin

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Actively transcribed genes

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Heterochromatin

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Inactive genes

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B form of DNA

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1. Biological form
2. Right handed helix
3. 10 bp/turn

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A form of DNA

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1. Right handed
2. 11 bp/turn
3. ssDNA
4. DNA:RNA
5. RNA:RNA

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Z form of DNA

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1. Left handed
2. 12 bp/turn
3. Active chromatin

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Endonucleases

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Cleave within the DNA strand.

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Exonucleases

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Cleave the ends of the DNA strand

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Importance of methylation in DNA synthesis

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Proofreading ability. The parental strand is methylated and the new strand is not methylated until it has been checked for errors.

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Repair mechanism with double strand DNA breaks

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1. Quick and dirty ligation of broken strands
2. In important genetic or modulary regions use the other chromosome as template.

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P53 role in repair

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Prevents cell replication before DNA repair has occurred.

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siRNA

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1. Defense mechanism in lower eukaryotes and plants.
2. Around 25 base pairs long
3. Have 5’ phosphates and 3’ hydroxyl groups and 2 to 3 nucleotide overhands on the 3’ end
4. All that helps with binding to RISC
5. siRNA is unwound by RISC activity and antisense strand is left to bind to target mRNA

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Dicer

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1. Family of RNase III enzymes
2. Recognize and process dsRNA into siRNA
3. Each dicer enzyme has an amino terminal helicase domain, 2 RNase III catalytic domains, dsRNA binding domain and a PAZ domain
3. Dicer is thought to act as a dimer of 2 enzymes – why?
We know that siRNAs are around 25 bp long
dsRNA cleavage is performed by the tandem RNase III catalytic domains - a single dicer enzyme will cleave dsRNA into 12-13 bp fragments
Put them together in a dimer, the 2 internal catalytic domains are made non-functional and the remaining catalytic domains are spaced far enough from each other to generate 25 bp fragments

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RISC

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1. RISC – RNA-induced silencing complex
2. Components of RISC are still unknown… but what’s is known so far:
2 RNA binding proteins – bind the siRNA
RNA/DNA Helicase – unwinding of the siRNA
Translation Initiation Factor – mutations of this component affect the initiation step
RNA-Dependent RNA Pol – may be a part of the RISC complex – may not – but it plays a role in the triggering and amplification of the silencing effect
Transmembrane Protein – seems to be the protein involved in the systemic spread of the RNAi… found in plants and so far in animals it’s only found in c.elegans

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Plasmid cloning vectors

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1. Plasmid contains origin of replication, antibiotic resistance gene, and polylinker region
2. new DNA with sticky ends is annealed to plasmid
3. Amplification occurs with antibiotic (the plasmid should be resistant) as the selection marker
4. You can prepare a genomic library in a plasmid vector

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Lambda phage cloning

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Similar to plasmid cloning but with large amounts of DNA

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Yeast artificial chromosomes

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Have the advantage of being eukaryotic and can hold large amounts of DNA>

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Genomic libraries

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Have all the DNA in the body including:
1. Introns
2. Promoters
3. Non-transcribed spacer DNA

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cDNA libraries

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Have only expressed post-modified genes in mRNA form.

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Complimentary endonucleases

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DNA is inserted into a cloning vector after cleavage with these that create a direction.

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2 methods to produce DNA probes

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1. Separate DNA strands and add radioactive dNTPs
2. Use polynucleotide kinase to transfer radioactive phosphate to 5' end of DNA

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Chemical Method (Maxam-Gilbert) of DNA sequencing

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1. 4 test tubes used
2. Chemical will attach to designated nucleotide and break the strand
3. Results in a bunch of different sized strands that are sequenced
4. DNA footprinting also uses chemicals to determine where specific proteins bind.

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Enzymatic method (Sanger's dedeoxy) of DNA sequencing

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1. Make 4 different vials and add a specific dideoxy "chain terminating" nucleotide into each vial
2. Results in stoppage in synthesis when these are encorporated
3. Anti-viral drugs use the same dideoxy method

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Automated Sequencing of DNA

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Uses fluorescent tags on dideoxy nucleotides that can be read by a machine as a certain color.

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Southern Blot

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1. Electrophoresis to separate different DNA fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids

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Northern Blot

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1. Electrophoresis to separate different RNA fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids

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Western Blot

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1. Electrophoresis to separate different protein fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids

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Restriction fragment length polymorphism

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Detects mutations in gene sequences by knowing the digestion patterns of certain endonucleases. You cleave the DNA and identify the digested pieces
1. Use southern blotting
2. Prenatal screening for sickle cell and phenylketonuria

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Polymerase Chain Reaction

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Used to make copies of genetic information
1. Heat to separate strands
2. Add primers to hybridize and add dNTPs
3. Heat and use Taq polymerase to transcribe
4. Repeat many cycles

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Variable number of tandem repeats

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Unique sequences outside of the coding regions of DNA that are amplified for identification purposes.

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Transcription methodology

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1. Synthesis of RNA from DNA templates
2. Uses RNA polymerase
3. Helicase and topo I for accuracy
4. Promoters, enhancers, repressors, and insulators for regulation

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Promoter

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DNA sequence required for initiating transcription; always orientation dependent.

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Enhancers

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Sequences that enhance rate of transcription. Generally position and orientation independent and can be large distances away.

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Repressors

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Work on promoters and decrease the activation of a gene. Often a long way away from the gene.

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Insulators

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Sequences on either side of a gene that insulate from the influence of adjacent genes.

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Coupled Transcription/Translation

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Occurs in prokaryotes because of the lack of nucleus.

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Uncoupled Transcription/Translation

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Occurs in eukaryotes because the nucleus is enclosed by membrane.

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Polycistronic

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One strand of Prokaryotic mRNA produces multiple proteins.

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Monocistronic

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One strand of eukaryotic mRNA produces one protein.

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Effect of methylation on transcription

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Methylation on promoters leads to inactivation of genes.

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Effect of acetylation on transcription

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Acetylation on histones leads to activation of genes.

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Eukaryotic mRNA processing

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1. 5' guanylmethyl cap
2. 3' polyadenine tail
3. Removal of introns and alternate and trans splicing

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Alternative splicing

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Ligates together different combinations of exons from the same gene.

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Trans-splicing

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Ligates together different combinations of exons from different genes.

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snRNP spliceosome

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Made of 7 subunits of snRNA and proteins that comes together to cleave out introns and splice together exons.

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Required things for translation

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Synthesis of proteins from mRNA template:
1. mRNA
2. Ribosomes
3. tRNAs
4. Amino acids
5. Protein factors
6. Energy (ATP, GTP)

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Two functions of tRNA

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1. Bind correct amino acid at 3' end through aminoacylsynthases
2. Bind triplet codon on mRNA using anti-condon segment

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Wobble hypothesis

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The third position in anti-codon pairs loosely leading to imperfect pairing and the necessity for less separate tRNAs. Increases rate of protein synthesis.

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Positioning of ribosome on mRNA in prokaryotes

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The 16S subunit binds 3' end of mRNA at a specific sequence and then reads and starts translation.

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Positioning of ribosome on mRNA in eukaryotes

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Ribosome starts at the 5' cap and scans for the start codon (AUG)

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Initiation

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1. Initiation factors aid small ribosomal subunit with initiator tRNA bound (AUG) to find start codon.
2. Large subunit binds and the next tRNA

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Elongation

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1. Each new amino acid gets transferred to growing chain.
2. Acceptor site - entry site for tRNA
3. Peptide site - place of peptide bond synthesis
4. Exit site - place of tRNA removal

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Termination

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When it reaches a stop codon a tRNA like release factor comes and binds to ribosome releasing protein.

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Polysomes

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Multiple ribosomes translating a single mRNA at once on RER surface or in cytosol.

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Post-translation

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Proteins are folded and undergo post-translational modifications

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Silent mutation

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The switched nucleotide codes for the same amino acid so the result protein is the same.

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Missense mutation

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The switched nucleotide codes for a different amino acid so the result protein is different.

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Nonsense mutation

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The switched nucleotide codes for a stop codon so the protein is truncated or nonfunctional.

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Addition/deletion mutations

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Addition or deletion of a single nucleotide resulting in a frame shift.

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Frame-shift mutation

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Changes the entire reading frame resulting in either a completely different protein or a shortened or nonexistant protein.

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Untranslated regions

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At the 5' and 3' end of mRNA confer stability and places for regulatory proteins to bind.

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Method of transferrin and aconitase activity.

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1. Iron binding to transferrin removes inhibition
2. Iron binding to aconitase induces degradation
3. In low iron environment - inhibits TR translation and stabilizes aconitase.

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Ferratin

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A protein responsible for storage of iron in a cell.

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Transferrin

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A surface receptor that brings iron into the cell.

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Recombinant proteins

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Use a plasmid to clone a desired gene into a bacteria, grow up bacteria, and purify protein for use as medication.

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Methods to make recombinant proteins

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1. Plasmid
2. Expression of recombinant genes in mammary glands so proteins are released in milk.

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Recombinant proteins used in medicine

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1. Tissue plasminogen activator
2. Blood clotting factors
3. Interleukins
4. Enzymes for research
5. Safer vaccines
6. Insulin

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Transgenic Mice

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Can be used to make disease models because of similarities between human and mouse genes
1. Gene replacement
2. Gene knock-out
3. Gene addition

Grow mouse stem cells in culture, alter DNA and then introduce them into early embryo, breeding these to get purebreds.

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Severe Combined Immune Deficiency

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Results from adenosine deaminase deficiency which causes an inability to make quickly dividing cells like B and T lymphocytes.

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Adenosine deaminase

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Needed for the break down of adenosine from food and for the turnover of nucleotides in the cell.

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Treatment for SCID

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Gene therapy:
1. Take stem cells from patient
2. Add the ADA gene using a viral vector
3. Introduce patient's cells back into system

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Long tandem repeat segments

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Used by viruses as location of placement into the genome.

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Cloning sequence when using a viral vector

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LTR-Enhancer-promoter-gene cDNA-poly(A) signal-LTR

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GLEEVAC

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Attacks a fusion protein made only in cancer cells of chronic myeloid leukemia. Prevents these from becoming cancerous until blood turns over and there is no longer cancer.

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BCR/ABL

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Fusion of chromsomes 23 and 9 result in this mutant gene that causes chronic myeloid leukemia.

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Methods of Oncogene Discovery

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Random genes from a cDNA library introduced into cells. Those that show overgrowth phenotype are oncogenes. Led to the discovery of Ras oncogene.

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Dicer in humans

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We have dicer and new research is looking at using it to chop up viral or cancer mRNA in human disease.

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Helicase

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Enzyme that separates DNA strands.

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Primase

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Enzyme that synthesizes RNA primers.

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Single stranded DNA binding proteins

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Keeps DNA protected and exposed for copying.

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DNA polymerase

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Multi-subunit enzyme that adds one nucleotide at a time to the 3'-OH of primer RNA or elongating DNA chain.

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Nucleosome

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250 bp of DNA covered in histones.

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Normal base to abnormal base mutations and repair

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Mutation: Caused by covelent bond formation of carcinogens or thymine dimerization which distort DNA helix.

Repair: Detected by DNA repair enzymes due to deformation of helix. Endonucleases cleave on either side of the damaged DNA and exonucleases cleave further along these nicks. DNA polymerase repairs and DNA ligase fuses the backbone.

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Endonuclease

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Enzyme that cleaves DNA or RNA in the middle.

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Exonuclease

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Enzyme that cleaves DNA or RNA from the end.

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Normal base to normal base mutations and repair

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Mutation: An incorrect amino acid is incorporated into a DNA strand.

Repair: Methylation occurs on the parent strand before DNA synthesis. The newly created strand has no methylation so the cell knows which one has the mutation on it when the base pairs do not match up. Mismatches are removed and repaired by DNA polymerase and ligase.

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Two ways to repair double stranded breaks in DNA

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1. Nonhomologous end joining in which the ends are simply ligated back together
2. Homologous end joining - copying process involved where the complete sequence is restored by copying from the second chromosome.

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p53

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Tumor suppressor protein that recognizes damaged DNA and leads to cell cycle arrest so that cells do not replicate damaged DNA.

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Self versus foreign macromolecules in humans

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Our immune system functions at the protein level, recognizing our own proteins from foreign proteins.

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Self defense for bacteria

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Restriction enzymes that degrade foreign DNA. They protect their own DNA by methylation at sites of action of restriction enzymes.

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Self defense for lower eukaryotes and plants

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siRNA which degrades foreign RNA potentially from viruses.

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Cloning

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Digest cellular DNA and plasmid DNA with a restriction enzyme, anneal, join with DNA ligase. Plasmid introduced into bacteria, copied, and purified.

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Anti-viral drugs

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Modified nucleotides called nucleosides are used. They incorporate into growing DNA chains and prevent further chain elongation. AZT.

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Introns

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Intervening sequences between exons. Few genes in lower eukaryotes. Most genes in higher eukaryotes.

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Editing

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Altering codons after transcription. Infrequent in humans; common in lower eukaryotes.

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Cap binding proteins

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Bind to the guanylmethyl cap and allow it to interact with the polyA tail to facilitate circularization of mRNA.

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PolyA binding proteins

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Bind to the poly A tail to stabilize mRNA and form a circle with cap binding proteins.

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Soluble protein translation

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Soluble proteins are translated on free-polyribosomes.

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Secreted protein translation

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Translated on the endoplasmic reticulum.

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3' untranslated regions

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Determine stability (halflife) of the mRNA.

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5' untranslated regions

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Regulates its own translation by binding molecules that can prevent translation machinery from being able to move.

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Exosome

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Degrades RNA. Several ribonucleases that work by removing polyA tail and removing cap structure allowing RNA to be degraded by exonucleases from 5' and 3' ends.

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Macrocytic anemia

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Results from deficiency in folic acid or vitamin B12. Causes problems with DNA synthesis which means while the cell is trying to replicate its DNA it is growing large.

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Microcytic anemia

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Results from deficiency in iron which leads to problems with hemoglobin synthesis resulting in tiny cells.