Biology 313: Exam 3

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Meselson and Stahl

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used labeled N markers to prove that DNA is passed semi-conservatively

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requirements for DNA synthesis

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-DNA polymerase : the synthesis enzyme complex.
-template (to be copied)
-primer with a free 3' hydroxyl group
-4 deoxynucleoside triphosphates (precursors)
-Magnesium ions Mg++ (co-factors for enzymes)
-ATP (energy source)

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direction of replication

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read: 3'-5'
synthesis: 5'-3' (added to 3' OH)

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phosphodiester bond

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formed during DNA synthesis

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Initiator protein

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binds to an initiation site (origin of replication) and separates DNA strands

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DNA gyrase

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Unwinds the supercoiling of DNA
-releases torque of DNA ahead of replication fork

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DNA helicase

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(a topoisomerase) opens the double helix in front of the replication fork

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Primase

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Synthesizes an RNA primer

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DNA polymerase 3

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-Extends the strand by addition to the 3’ end of the primer
-Proofreads the new DNA strand (using its 3' to 5' exonuclease activity)

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single-stranded binding proteins (SSBs)

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Stabilize the single strands to prevent secondary structures

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DNA polymerase 1

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removes RNA primers and replaces them w/ DNA

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DNA ligase

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joins Okasaki fragments by sealing the nicks in the sugar-phosphate of newly synthesized DNA

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speed of DNA polymerase

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eukaryotes: 50nt per second
prokaryotes: 1000nt per second

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telomeres

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GC rich ends of chromosomes, protected from degradation by telomerase

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telomerase

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present in single-celled organism, germ cells, early embryonic cells, and bone marrow cells/intestinal cells

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Holliday model

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predicts noncrossover or crossover recombinant DNA, depending on whether cleavage is in the horizontal or vertical plane

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gene conversion

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process of nonreciprocal genetic exchange that can produce abnormal ratios of gametes

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Transcription: DNA --> RNA

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one strand used
read: 3' ->5'
synthesized 5' -> 3'

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promoter region

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transcription initiates

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prokaryotic promoter

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-10 (Pribnow) = position the polymerase
-35 = improves polymerase binding efficiency

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prokaryotic RNA polymerase

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5 proteins (core) + sigma factor = holoenzyme

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sigma factor

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must be added to RNA polymerase in order to bind; released once transcription begins

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consensus sequences

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sequences that possess considerable similarity (present in most organisms w/ similar functions)

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bacterial termination

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inverted repeats form a hairpin loop followed by a string of uracils
-Rho-dependent: able to cause termination only in presen

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Rho-dependent termination

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rho factor necessary (moves along RNA when polymerase pauses @ termination sequence)

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Rho-independent termination

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1. contains inverted repeats (hairpin)
2. repeated adenine (and therefore uracil)

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basal transcription apparatus

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RNA polymerase + general transcription factors (proteins

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RNA polymerase 2

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transcribes genes that encodes promoters

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core promoter

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(-35) TF2B recognition element
(-25) TATA box = basal transcription app. binds to this core promoter site

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regulatory promoter

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located immediately upstream of core; binds transcriptional activator proteins (helps promote transcription)

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Eukaryotic initiation

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-TFIID binds core promoter (with help of TataBP)
-transcription factors & RNA polymerase II bind promoter
-enhancer proteins bind to complex
Transcription begins

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Eukaryotic termination

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(Transcription termination signals are different for each polymerase)

Pol II synthesizes beyond the gene.
RNA is cleaved
Rat1 attaches to RNA (endonuclease action degrades RNA)
Pol releases DNA

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introns

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removed in post-transcriptional processing
-often code for more bp than exons

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exons

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part of gene that exits the nucleus

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5’- Capping

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addition of m7G and methylation of newly formed mRNA in Eukaryotes

-7-methylguanine added in a 5’ <–> 5’ linkage

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poly-A (Polyadenylation) tail

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addition of 50-250 A’s to 3’ end by Poly(A) polymerase

-Transcript is cleaved at cleavage site (consensus sequence: AAUAAA; 11-30 nucleotides upstream)
-polyA sequence added

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splicing

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removing introns in nucleus

-requires:
1. 5' splice site
2. 3' splice site
3. adenine branch point

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lariat

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loop formed after 5' splices, formed b/t 5' phosphate and 2' OH of A branch

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degenerate code

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More than one triplet codes for one amino acid

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initiation codon

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methionine AUG

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termination codons

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UGA, UAA, UAG

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Amino acids

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-Amino group
-Carboxyl group
-Radical group

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Peptide bonds

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forms between amino acid

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trans splicing

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combining exons from two or more different pre-RNAs

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alternative processing

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splicing: same pre-mRNA can be spliced in more than one way to yield multiple mRNAs

multiple 3' cleavage sites: two or more sites for cleavage are present on pre-mRNA

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modified bases

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unique to tRNA; altered nucleotides

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cloverleaf tRNA structure

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four arms:
1 acceptor
2 T?C (modified bases)
3 anticodon
4 DHU (modified bases)

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rRNA

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eukaryotes:

prokaryotes:

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protein structure

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primary: amino acid sequence
secondary: B pleated sheet and a helix
tertiary structure: overall, 3D shape of protein
quaternary structure: 2+ polypeptide chains

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sense codons

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code for amino acids (61/64)

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wobble

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cause for degenerate code; flexibility of third codon

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tRNA charging

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aminoacyl-tRNA synthesases pair amino acids with tRNA (based on nucleotide sequences)

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Prokaryotic ribosomes

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70S

*30S subunit: 16S rRNA + 20 proteins
*50S subunit: 5S and 23S rRNA + 34 proteins

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Eukaryotic Ribosome

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80S

* 40S small subunit: 18S rRNA + ~ 30 proteins
* 60S large subunit: 5S, 5.8S, 28S rRNA’s + ~ 50 proteins

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initiation of translation

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shine-dalgarno sequence matches to small subunit

-IF attach
-large subunit attaches
-IF released

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IF3

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binds to small subunit, preventing large subunit from binding

-allows small subunit to bind to mRNA

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3 sites on ribosomes

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(right to left)
a: aminoacyl
p: peptidyl
e: exit

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Elongation Factors

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deliver, regenerate GTP

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peptidyl transferase

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part of ribosome; creates peptide bond b/t amino acids in A and P site

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EF-Tu

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binds GTP and charged tRNA; delivers charged tRNA to A site

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polyribosome

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multiple ribosomes attach to 5' site and begin synthesis on one mRNA

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Multiple Levels of Gene Regulation

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1. alteration of structure
2. transcription
3. mRNA processing
4. RNA stability
5. translation
6. posttranslational modification

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negative vs positive control

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negative: regulatory protein is a repressor

positive: regulatory protein is an activator

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inducible vs repressible

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inducible: normally off, must be turned on

repressible operons: normally on, must be turned off

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negative inducible

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reg. protein is a repressor (readily binds to operator site)

-another molecule (inducer) allosterically changes repressor to begin translation

(inducible often control proteins that break down molecules)

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negative repressible

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reg. protein is inactive repressor

-must have another molecule (corepressor) to prevent translation

(repressible control of proteins that carry out biosynthesis of molecules needed in cell)

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histone modification

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acetylation: stimulates transcription; destabilize structure

methylation: depending on amino acid, may stimulate or repress

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DNA methylation

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cytosine bases (usually adjacent to guanine (CpG islands))

-repression

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activators

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usually contain DNA-binding motif
-helix-turn-helix
-zinc finger
-leucine zipper

*interact w/ core promoter (several sites for different activators)

*may also open chromatin

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chromatin remodeling complexes

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bind directly to particular sites on DNA and reposition the nucleosomes, allowing transcription factors to bind to promoters

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phosphorylation

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maintains histone structure

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enhancer

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affect transcription at distant promoter

-activator proteins bind to enhancer
-create loop to transcription apparatus, help stabilize
-essential to transcription initiation

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transcription complex

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TBP: binds TATA box
TAF = TATA Associated factors

RNA polymerase II
(Associated polymerase factors: TFIF, TFIIE, TFIIH, TFIIJ )

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Gene-splicing in Drosophilia

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depends on X:A ratio -> determines whether Sxl (sex lethal) protein is produced

Sxl proteins (presences) allows for correct splicing of tra pre-mRNA

-Tra protein (presence) determines sex-specific splicing of dsx pre-mRNA

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dicer

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cleaves and processes double-stranded RNA to produce siRNAs or miRNAs

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RISC

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RNA-dinduced silencing complex

(proteins + siRNAs and miRNA)

regulate through:
1. RNA cleavage ("slice")
2. inhibition of translation
3. transcriptional silencing (alteration chromatin structure)
4. slicer-independent RNA degradation

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gene mutation

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INHERITABLE change in DNA sequence of one gene

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somatic mutation

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non inheritable;

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Changes in the coding region:

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-Changes in amino acid sequence
-No change in amino acid sequence (silent)
-Replace an amino acid with a similar amino acid (neutral)

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Transition

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purine is replaced by a purine (A<-> G)
pyrimidine is replaced by a pyrimidine (T<-> C)

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Transversion

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-purine is replaced by a pyrimidine (A<->C)
-pyrimidine is replaced by a purine (T<->G)

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Trinucleotide repeats

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Some genes have tandem repeats of trinucleotide sequences. Insertion mutations increase the number of copies.

Example: Fragile X
-occurs in germ cells
-repeated ends pair together to form hairpins
-may cause visible chromosome changes

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Missense mutations

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change single amino acid

-functional OR nonfunctional products

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Nonsense mutation

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creates stop codon

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Silent mutations

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creates codon that codes for the same amino acid

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reverse mutation

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convert original mutation back to wild type

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suppressor mutations

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act in a way that reverts the original mutation and produces wild phenotype

-example one change in codon

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Tautomeric shifts of bases

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-Changes in base hydrogen bonding
-Changes in base pairing
-made permanent through replication

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Deamination

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loss of amino group from cytosine

-creates U or T

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base analogs

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Compounds with similar structure to bases, have high frequency of tautomeric shifts

-cause either transitions OR transversions

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Chemical mutagenic agents

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Alkylating agents, EMS
-Deaminating agents, Nitrous acid
-Hydroxylating agents, -Hydroxylamine

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UV radiation

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produce thiamine dimers

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Ionizing Radiation

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deamination, depurination, chromosome breaks.
(no safe dose of ionizing radiation)

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DNA repair

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Cells have several mechanisms to repair DNA lesions

Photoreactivation

Excision repair

SOS repair (error prone repair)

Double strand repair

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exicision repair

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Mismatched bases are removed and new DNA synthesized.
Methylation patterns are key to recognition of the template.