Wednesday, June 13, 2012

Biological Science 2 (Molecular Biology: DNA and Protein Synthesis)


Day 4:


Really lost? This book would be helpful for people who haven't taken genetics, some of it is available free. I looked through it. It is very easy to understand, but accurate enough for the MCAT.

DNA Structure and Function
A. DNA Structure and Function
DNA is two strands in opposite directions next to each other (due to hydrogen bonds), twisted into a double helix. DNA codes for the living material that will be built, it regulates living cells, programs death of other cells, turns genes on or off, carries traits through generations, results in survival of the species through evolution. "Junk DNA" may even carry the memories of ancestors...

DNA Reveiw from the National Science Foundation

1. Double-Helix Structure
DNA is a double-helix. The sugar is the 3' carbon and the phosphate is the 5' carbon. RNA and DNA polymerase read from the 3' end and made from the 5' end. The double-helix can unzip like a zipper then be copied from the 3' side.

2. DNA Composition (Purine and Pyrimidine Bases, Deoxyribose, Phosphate)
DNA has Purines (adenine and guanine, large, 5-membered ring fused to 6-membered ring)
DNA has Pyrimidines (thymine and cytosine, small 6-membered rings)
Deoxyribose is a 5 carbon sugar
Phosphate on the 5' covalently bonds with sugar on the 3'

3. Base-Pairing Specificity, Concept of Complementarity
DNA has two pairs (CG stronger):
AT (2 hydrogen bonds): 

GC (3 hydrogen bonds):

4. Function in Transmission of Genetic Information
DNA is a code for production of tissue (proteins) and control of cells and genes. DNA is the basis for all genetic information, as it's order of base pairs is the blueprint for all structures built by the body and all control of use of genes as well.

B. DNA Replication
From Barron's MCAT Test Prep Book 12th edition (thanks to Hugo Seibel and friends): 
DNA unzips itself, the 3' gets new neucleotides bonded to it one at a time. 5' is written first.
Eeukaryotes use DNA polymerase alpha/delta (lagging/leading). 
Prokaryotes use DNA polymerase III

1. Mechanism of Replication (Separation of Strands, Specific Coupling of Free Nucleic 
Acids, DNA Polymerase, RNA Primer Required)
DNA Strands Separate:
      DNA gyrase (class II  topoisomerase) uncoils DNA ahead of the replication fork
        Helicase is responcible for unwinding DNA at the replication fork
      Single-Stand Binding Protein stabilizes single-strand DNA by binding to it 

DNA Gets Primed:
      Primase puts an RNA primer onto the DNA 
      DNA Polymerase starts making DNA complementary to the unwound DNA (AT-GC)
RNA Primers Replaced:
      DNA Repair Complex with DNA Polymerase I Replaces the RNA Primer (leading)
      DNA Ligase connects Okazaki fragments (lagging) 

2. Semiconservative Nature of Replication
New DNA is made of one old (conservation) and one new strand.

C. Repair of DNA (Didn't you proof-read this sequence DNA polymerase?)
Repair is a normal process. Different errors or amounts of errors lead to different repairs.

1. Repair During Replication
DNA polymerase proof-reads 3'-5' and removes and replaces 5'-3' when there are errors.

2. Repair of Mutations

Photoreactivation (Ex. Pyrimidine dimers):
      Pyrimidine dimers (TT,CC) produced by UV radiation are repaired by DNA Photolyase  
      AKA Photoreactivating Enzyme using visible light energy to remove the covalent bond 
      caused by UV radiation (not in humans).

Base Excision Repair:
      Some single damaged bases (especially uracil) are removed by a glycosylase enzyme,  
      then the sugar and phosphate and surrounding nucleotides are removed.

Nucleotide Excision Repair (Ex. Thymine Dimers):
      One strand is damaged and the other is not, methylation of the older strand determines   
      which stand is used to rebuild the damaged strand. Mismatch Repair works the same 
      way, but it is triggered by mismatch (not damage).

Nick Translation (Ex. RNA Primer Replacement)
      The proof-reading (exonuclease activity) of polymerase.

D. Recombinant DNA Techniques
1. Restriction Enzymes (Restriction Endonucleases)
Restriction enzymes cut double stranded DNA at palindrome sequences (the same both directions), causing restriction fragments which may have sticky ends (1:5 to hybridize) or blunt ends (3:3 can not hybridize).

2. Hybridization (Annealing)

  • DNA strands base pair with each other. 
  • DNA probes hybridize onto DNA fragments containing a target sequence (Southern Blotting).
  • Sticky ends from a restriction fragment of a gene base pair with the same sticky ends on a plasmid (gene cloning).

Day 5:

3. Gene Cloning 

  • Genes are cut into (sticky ends) with a restriction enzyme
  • Plasmid rings are also cut open with the same restriction enzyme (leaving sticky ends)
  • Gene peices glob onto the cut open plasmids (called hybridizing)
  • DNA ligase seals the Recombinant Plasmid (hybridized ring of plasmid and gene pieces)
  • The recombinant plasmid is inserted into a bacteria (more or less rape of a bacteria)
  • Replication inside the bacteria occurs creating more of the gene                                        
The plamsid needs to have a restriction site to cut it open, plasmids can be given antibiotic genes to kill off competing bacteria without harming the ones making genes, plasmids replicate independently of the bacteria's genomic DNA.

Gene Cloning Review (MC Graw Hill) 

4. PCR (Polymerase Chain Reaction) 
Instead of cloning, a chain reaction can copy a gene billions of times, by using a lot of primers to out-compete DNA strands with extra length, a relatively pure batch of genes are produced.

  • Denaturation seperates DNA by overcoming the weak hydrogen bonds (90 °C)
  • Annealing (at cooler temperature) primers (in excess) anneal to single stranded DNA
  • Elongation using heat stable polymerase to extend primers                                  (Repetition of the cycle makes  2n  more DNA, after n number of cycles)  -

Weird Vocabulary: Anneal- To subject (glass or metal) to a process of heating and slow cooling in order to toughen and reduce brittleness. Or, to strengthen or harden. Ex. I annealed the gene continuously to make billions of copies for show and tell.

Introduction to PCR (thanks DR. Zeeshan)

Low quality pop music is needed to drive the reaction (not really).

Real-time Polymerase Chain Reaction Video
It's fun to see a non-abstract video sometimes.

Polymerase Chain Reaction Review Song (Seriously)


Protein synthesis involves RNA. DNA can control protein synthesis by controlling the RNA, but DNA has to be converted to RNA (switching out thymine with uracil and deoxiribose with ribose) in order to make protein. 

Protein Synthesis (Ribosomes Translating mRNA into Protein)

A. Genetic Code
DNA Replication (the DNA is doubled if the cell is going to divide). 

1. Typical information flow (DNA → RNA → protein)

RNA Transcription (if genes are needed, extra copies are made from the DNA in RNA form) Like writing a recipe a friend asked for from a Spanish cook book in your house and giving them a sheet of paper with the English version of just the recipe they want (with everything unnecessary removed). In that case, writing the recipe down from Spanish (DNA) to English (RNA) would be called RNA Transcription. The paper would be a mRNA molecule. The paper would have a sticker with your friends name reminding you to take it out of your house and deliver it to your friend, the sticker would be an export signal protein. The part where you remove unnecessary information from what you include in your friend's copy is called Processing. When the paper gets taken out of your house (the nucleus) it is called Transport. When your friend cooks a meal with your recipe it is called RNA Translation. Your friend is a ribosome by the way. DNA is the cookbook, RNA is the individual meal recipe and protein is the cooked meal. The anticodons are the squiggly green things with colored tick tacks on them in the above video. They are also green the video below.

Protein Synthesis (Ribosomes Translating mRNA into Protein)

DNA -> RNA -> Protein

2. Codon–anticodon relationship, degenerate code
Codons are like 3 letters (3 nucleotides) that give a signal for a particular amino acid.
Like a user name that can only be 3 letters long and only 4 letter were usable CTAG...
Codons are continuous (if you wanted to write about a group, list all the user names with no space EX. MOMDADBROMYF mom, dad, brother, myself).
Codons are non-overlapping Ex. MOMMYF not MOMYF, the M or any letter can not count for more then one word at a time.
Codons are degenerate because more then one codon codes for a single amino acid Ex. Glutamine had a user name CAA, but forgot and made another user name CAG, Glutamine can log in as either one now... that's better then Leucine, who has six user names, degenerate!

Condons are not the same a anticodons. Codons are 3 nucleotides and anticodons are 3 nucleotides that pair up correctly or match. A codon CAA, has an anticodon GUU. Since, A always pairs with U and C always pairs with G. Ex. If the codon is boots, dress, high heels the anticodon is cowboy hat, high heels, dress. Or if the codon is peanut butter, peas, carrots the anticodon is jelly, carrots peas. The anticodon is located on the tRNA. One side or tRNA has an amino acid and one side has an anticodon. During translation, codons pair with their particular anticodons and the amino acids end up lining up since they are attached and they end up bonding to the amino acids next to them too (like the spouses of two best friends being forced to get to know each other because they are always dragged to the same events, Ex. Glutamic acid says: Hi Lysine, your anticodon wanted to see Twilight too? Lets just form Ribonuclease Protein with the other 122 amino acids while we are here.)...

Ribonuclease One of the Simplest Proteins

3. Missense and nonsense codons
Missence Codons: Mutated codons result in different amino acids. "Excuse me, I wanted        -       -                      Asparagine not Aspartic Acid."
Nonsencse Codons: Result in stop codons or non-amino acids. "Excuse me, I wanted                                -                               Histidine not Sierra Mist."

4. Initiation and termination codons (function, codon sequences)
Initiation: (AUG
All u guys... listen up
Terminaion (UAG, UGA, UAA
I'm done listening (u are great)/(ur gayness annoys me)/(u are angry right now) 

Initiation codons bind with tRNA.
Termination codons trigger release factors not tRNA.

B. Transcription
Making RNA copies of pieces of DNA in the nucleus. 

  • Initiation: the RNA Polymerase sees a bookmark (promoter) that lets it know where to start copying (promoter recognition). The Book (double stranded DNA) is opened. 
  • Elongation: copies are made of the book pages, but they are black and white (RNA) not color (DNA), they are still readable.
  • Termination: when the end of the needed chapter is reached copying is done. 

Transcription of DNA into RNA (Staring mRNA)

1. mRNA composition and structure (RNA nucleotides, 5′ cap, poly-A tail)
mRNA (messenger RNA) is made of neucleotides (uracil, cytosine, adenine and guanine) during transcription (in the nucleus) and used for translation (in the cytoplasm). The 5' cap and poly-A tail prevent exonuclease degradation at the 5' and 3' end respectively (only in Eukaryotes). 

2. tRNA and rRNA composition and structure (e.g., RNA nucleotides)
tRNA (transfer RNA) and rRNA (ribosomal RNA) are made during transcription (brothers of mRNA), but they don't get translated into protein like mRNA does. tRNA brings the amino acid to mRNA and rRNA forms the ribosome. The tRNA has a clover shape, the amino acid on a tRNA is attached at the 3'OH by an ester linkage, on one side, on the opposite side has the anticodon. The tRNA that looses its amino acid leaves at the E site of the complex. The rRNA of the large ribosomal subunit catalyzed peptide bond formation.

3. Mechanism of transcription (RNA polymerase, promoters, primer not required)

  • Chain Initiation: RNA polymerase latches onto the promoter (TATA box) of the double stranded DNA opening up the complex.
  • Chain Elongation: Nucleoside Triphosphates (AUGCs) add correspondingly to the present DNA. No primer is used (RNA polymerase is like 2 in one paint and primer). RNA is made in the 5' to 3' direction (like DNA). 
  • Chain Termination: Sequences cause a stem-loop structure that causes the RNA to slip off (intrinsic termination) or Rho (ρ) dependent termination occurs due to a protein called the ρ factor that bumps off the polymerase. -                                       

C. Translation
RNA is when mRNA is used to make proteins. 
tRNA get amino acids when enzymes called aminoacytl-tRNA synthetases attach the correct amino acids onto them (ATP required), then the tRNA bring the amino acid (attached to it) to a ribosome to connect the amino acid to a chain of aminoacids coded for by the mRNA.

  • Chain Initiation: An initiation complex including mRNA, initiator tRNA (fmet) and a ribosome is formed by initiation factors and GTP around an initiation codon (AUG) following a Shine-Dalgarno or Kozak sequence (in pokaryotes or eukaryotes respectively).
  • Chain Elongation: protein is made from the N terminus to C terminus. mRNA are read 5' to 3' (opposite of DNA). Binding occurs between the chain of amino acids and tRNA's amino acid at the A site of the tRNA (GTP and elongation factor required). Peptidyl transfer occurs at the P site by the whole chain moving onto the A site of the tRNA. The tRNA in the P site gets kicked of the E site. The codon that was in the A site is now in the P site (translocation) and the A site is open for binding to a new aminoacyl-tRNA to a new codon (GTP and elongation factor required).  
  • Chain Termination: a stop codon (UAG, UGA, UAA) causes release factors bound to GTP to block the A site. The chain gets cut off at the P site, falls off and the translation complex falls apart.

1. Roles of mRNA, tRNA, and rRNA; RNA base-pairing specificity
Imagine a smart middle brother, a dumber, yet diplomatic big brother and many other dumb little brothers all trying to manufactur drugs together. The smart brother (medium sized mRNA) knows how to make drugs, but doesn't want to do the leg work. The diplomatic, big brother (large rRNA, the ribosome) gets together with his smart brother to get the information and also gets his dumb little brothers (small tRNA) to bring him everything needed to make the drugs. So the big brother (ribosome) oversees production (by catalyzing petptide bonds), but the recipe is provided by the smart brother (mRNA). The little brothers (tRNA) are only concered with bringing ingredients (nucleotides) and then leaving to find bring more ingredients. RNA always pairs adenine with uracil and guanine with thymine (adenine and thymine broke up, but guanine and cytosine are still together).

2. Role and structure of ribosomes
Ribosomes catalyze protein synthesis by using tRNA to bring amino acids and mRNA to give the proper sequence. 

The ribosome helps to form peptide bonds (in the large subunit) by adding new amino acids to the existing chain at the P site. The whole chain moves to the tRNA to attach to the aminoacyl-tRNA in the A site (Hey tRNA, do you want to move over here to my P site? I'm hundreds of amino acids bigger then you. No, you can move over here to my A site). The ribosome has a large and small subunit (like an underachieving snowman). The small unit recognizes mRNA (binds to the Shine-Dalgarno sequence or Kozak sequence in eukaryotes). The large and small units come together to sandwitch the mRNA and tRNA. Who wants a 
mRNA and tRNA sandwitch? It has lots of protein...

1 comment:

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