Molecular Final - Dna (Nucleic Acids)

Front 1

Central Dogma of biology

Back 1

-DNA replication (transcription + translation)

-DNA - RNA - Protein

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Two kinds of nucleic acids

Back 2

-Differ from eachother in _ and _

-Third macromolecular component is _

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James Watson and Francis Crick introduced what....

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double helical model for DNA

-used info from other scientists (Rosalind FRanklin + Linus Pauling)

-strands = antiparallel

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Chargaffs rule

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ratios between bases (proportion of A = T, C = G)

-pyrimidine binds with purine -> reason for equal ratios

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Evidence about DNA structure

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Chemistry: nitrogenous bases

Biology: ratios between bases

Physics: X-ray crystallography (Rosalind Franklin)

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Deductions based of Franklin's X-Ray diffraction

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-DNA = helical

-width of helix, spacing of nitrogenous bases

-nitrogenous bases on inside (relatively hydrophobic)

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Types of bonds in DNA

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-phosphodiester bonds

-hydrogen-bond

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Correct model for DNA replication, other possibilities

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-semiconservative model

Other theories:

-conservative

-dispersive

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Experiment for Semiconservative model

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1. Bacteria + heavy isotope

2. Bacteria transferred to medium with lighter isotope

3. DNA sample centrifuged after 1st replication

4. DNA centrifuged after second replication

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DNA replication

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-2 strands are complementary

-1 serve = template

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Bacterial DNA replication

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-1 circular chromosome

-Origin of replication

-template strand and new strand

-replication bubble - replication fork

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Leading vs. Lagging strand

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-leading - synthesized continuously, moving toward the replication fork

-lagging - synthesized as a series of segments = Okazaki fragments

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DNA polymerase

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-catalyze replication

-cannot initiate -> require primer

so, short stretch of RNA synthesized by primase using parental as template

-Adds nucleotides ONLY to he free 3' end of growing DNA strand

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What direction does the new strand elongate in?

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elongates in the 5' to 3' direction

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Role of DNA helicase

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-unwinds the parental double helix

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SSB

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Single Strand Binding Protein

-keeps unwound strands in an extended form for replication

-avoids hairpin structures

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Supercoiling of unwound DNA

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replication fork spins

-swivel - reduces positive supercoiling in front of fork

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DNA topoisomerase 1

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-covalently attaches to DNA phosphate - breaks phosphodiester linkage

-energy from breakage is stored - reversible reaction (reformation regenerates helix)

-now strands can rotate

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Requirements of DNA polymerases

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-primer DNA with free 3'-OH

-template DNA

-substrates: dNTP's

-Magnesium

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What are some enzymes crucial to replication? What happen without?

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-Helicases: untwist double helix at rep. forks

-SSB proteins: bind/stabilize

-Topoisomerase: corrects "overwinding", breaks, swivels and rejoins DNA strands

-Primase: makes the primer

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Lagging strand fragments

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-each okazaki fragment - needs its own primer

After fragments are synthesized:

-DNA polymerase replaces RNA primer with DNA

-DNA ligase joins the sugar phosphate backbones into continuous strand

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What proofreads the DNA? What happens when it misses?

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DNA polymerase

-other enzymes correct errors in base pairing (mismatch repair)

-nuclease cuts out and replaces damaged bits (nucleotide excision repair)

-DNA ligase rebuilds the backbone whenever DNA is cut for nucleotide replication

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What is the evolutionary significance of DNA Nucleotides

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-sequence changes/mistakes may become permanent/get passed on to future generations

-mutations -> source of genetic variation

-leads to natural selection

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Progressive shortening of DNA why? Why aren't prokaryotes affected by this?

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-can't complete the 5' ends, repeated rounds of replication = shorter and shorter molecules w/ uneven ends

-not a problem for prokaryotes = circular chromosomes

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Telomeres, role and anticancer properties?

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-special sequences at end of Euk. chromosome

-postpone erosion near ends

-shortening of telomeres = aging

-limit # cell divisions (cancerous growth)

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Telomerase

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catalyzes the lengthening of telomeres in germ cells

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What is associated with premature aging?

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-short telomeres

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Transcription

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-template strand - RNA transcript (mRNA)

-transcription unit = stretch of DNA that's transcribed

-Promoter = sequence that signals the end of transcription (in bacteria)

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Stages of transcription

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1. Initiation

2. Elongation

3. Termination

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Translation

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-synthesis of polypeptide using mRNA info

-occurs on ribosomes

-requires ribosomal RNA (rRNA)

-requires transfer RNA (tRNA)

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Promoters

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signal the transcriptional start point on DNA

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Transcription factors

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mediate binding of RNA polymerase and the initiation of transcription

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TATA box

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crucial in forming the initiation complex in eukaryotes

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Termination of Transcription - bacteria vs. eukaryotes

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Bacteria: polymerase stops transcription at the end of the terminator, mRNA is translated into protein

Eukaryotes: RNA polymerase 2 transcribes polyadenylation signal sequence before being released

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pre-mRNA molecule modifications, why?

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-5' end gets modified nucleotide 5' cap

-3' end gets a poly-A tail

Modifications

-facilitate the export of mRNA to the cytoplasm

-protect mRNA from hydrolytic enzymes

-Help ribosomes attach to the 5' end

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RNA splicing

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Introns: Non-coding stretches

Exons: usually translated into amino acid sequences/are expressed

RNA splicing - removes introns, joins exons - create mRNA molecule with continuous coding sequence

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The functiona and evolutionary importance of introns

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1. some introns contain sequences that may regulate gene expression
2. alternative RNA splicing - some genes encode more than one kind of polypeptide

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Why can organisms produce a higher number of proteins than their number of genes?

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-depending on which segments are treated as exons during splicing, different sequences produce proteins

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Codon

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-three nucleotides long

-codes for an amino acid

-codons of gene -> transcribed into complementary non-overlapping codons of mRNA

-must be read in the correct reading frame

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What are the two required steps in translation?

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1. a correct match between tRNA and an amino acid

2. a correct match between the tRNA anticodon and an mRNA codon

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tRNA structure and function

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-small single RNA

-clover leaf

-ends of tRNA are not identical

-Wobble: flexible pairing at the third base of a codon allows some tRNAs to bind to more than one codon

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What is the role of aminoacyl-tRNA synthase?

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ensures correct match

-by joining specific amino acid to specific tRNA

-requires energy

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Ribosomes

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-two subunits (large and small)
-protein + ribosomal RNA (rRNA)
-facilitate coupling of tRNA codons in protein synthesis
-3 sites (E,P,A)

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3 Binding sites in Ribosome for tRNA

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1. A site (initial): holds tRNA that carries Amino acid being added

2. P site (central): holds tRNA that carries growing peptide chain

3. E site (exit site): where discharged tRNA's exit

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What is the result of having many ribosomes translating RNA at the same time? Advantages?

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-form polyribosome = polysome

-enables cell to make copies of polypeptide V quickly

-found in both bacteria and eukaryotic cells

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Free vs. Bound ribosomes

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Free - in cytosol: synthesize proteins that function in cytosol

Bound - attached to the ER: make proteins of the endomembrane system and proteins secreted from the cell

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What will cause the synthesis not to be in cytosol from start to finish?

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polypeptide begins in the cytosol and finishes there unless the polypeptide signals ribosome to attach to the ER

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Initiation of translation

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1. small ribosomal - binds with mRNA and the initiator tRNA

2. small subunit moves along mRNA until start codon

3. initiation factors - bring in large ribosomal subunit that completes the translation initiation complex

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The 3 steps of Elongation

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1. codon recognition

2. peptide bond formation at the C-terminus

3. translocation

-requires energy + elongation factors

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Termination of translation (4)

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1. stop codon in the mRNA reaches A site
2. release factor
3. addition of water molecule
4. polypeptide released, translation assembly comes apart

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How does the polypeptide become a functional protein?

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-coils - folds into 3D shape

-chaperonin protein helps it fold correctly

-may also require post-translational modification (adding, removal, cleavage, combination)

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How do proteins get targetted to the ER?

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-polypeptide gets marked by attaching signal protein

-signal recognition particle (SRP) binds to the signal peptide

-the SRP brings the signal peptide and its ribosomes to the ER

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Types of RNA molecules (5)

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-Messenger (carry genetic info)

-Transfer (adaptors between a.a and codons)

-Ribosomal (structural/catalytic component of ribosomes)

-Small nuclear (structural components of spliceosomes)

-Micro (block expression of complementary mRNAs)

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Mutations

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-changes in genetic material

-spontaneous

-caused by physical/chemical agents (mutagens)

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Point mutation:

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1 base pair

-Nucleotide-pair substitutions

-Nucleotide-pair insertions/deletions

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Substitution mutations

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-nucleotide-pair substitution

-silent mutation

-missense - code, but not for correct a.a.

-nonsense - change to stop codon

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Frameshift mutation

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-result of insertions or deletions

-additions/losses of nucleotide pairs

-may alter reading frame