cell bio unit 3

experiment 1: there is a transforming factor

experiment 1: there is a transforming factor

Frederick Griffith, worked with bacteria (streptococcus pneumonia) did 4 trials with mice, smooth strain kills, rough strain does not, boiled smooth strain does not, rough and smooth kills

experiment 1 conclusion

some molecules from smooth strain transferred to rough strain to make it pathogenic

experiment 2: DNA is the transforming factor

Alfred Hershey, Martha Chase, worked with bacteria, bacteriophage viruses, viruses have labeled proteins or DNA, allow viruses to infect bacteria, agitate to separate, centrifuge to separate by weight, measure radioactivity of top and bottom

experiment 2 conclusion

DNA is the transforming factor

experiment 3: Chargaff’s Base-Pairing Rules

Erwin Chargaff, works with nitrogenous bases of DNA, can quantitate A vs G vs C vs T

experiment 3 conclusion

different species have different concentrations of A/C/G/T, and certain bases were always approximately the same (A and T, C and G)

experiment 4: DNA is symmetrical in structure

Rosalind Franklin, x ray diffraction of crystal of DNA

experiment 5: The double helix

James Watson, Francis Crick, biochemists, found strands anti paralell, strands joined in center by H bonds b/w nitrogenous bases, (A and T 2), (C and G 3)

conservative method

the original 2 strands of DNA end up back together

semi conservative method

new cells get 1 original strand and 1 new strand

dispersive method

each cell has mix of old and new in chimeric form

experiment with Meselson and Stahl

work with bacteria, grow intially radioactive N15 media, switch over ot N14 media, new DNA N14, old DNA N15, then isolated DNA and centrifuge to separate by weight, see where radioactivity is

conclusion for experiment with Meselson and Stahl

semi conservative is how DNA replicates

goal of DNA replication

build copy of entire double stranded DNA genome as cell gets ready to multiply

DNA polymerase

key enzyme when replication DNA, adds new deoxyribonucleotides to 3’ end of growing nucleic strand (always add to 3’) 2 main types, requires template to guide them

DNA polymerase 1

fixes errors that they created, 3’ to 5’ exonuclease activity, also has 5’ to 3’ exonuclease activity

DNA polymerase 2

only can fix error that they create with 3’ to 5’ exonuclease activity

exonuclease activity

ability of DNA polymerase to remove nucleotides that have been incorrectly incorporated during DNA synthesis

molecular process of DNA replication

3 steps: initiate DNA replication, continue unwinding the double stranded DNA, lay down RNA primer to allow DNA polymerase a place to start primase

step 1: initiate DNA replication

origin of replication initiation (ori) proteins recognize and bind to ori site in double stranded DNA, ori proteins aggregate and force double stranded DNA to open and expose single stranded DNA template, single stranded proteins bind to exposed nitrogenous bases to keep strands from rejoining

ori proteins

origin of replication proteins

step 2: continue unwinding the double stranded DNA helix

helicase and topoisomerase used, both move away from origin of replication

helicase

enzyme that sits at replication forks, and moves away from origin of replication breaking H bonds b/w base pairs

topoisomerase

acts ahead of the helicase, moving in same direction, relieves tension that helicase causes in the unwinding, breaks one strand of DNA, unravels it, one torn by passing around other strand, then reforms the bond

step 3: lay down RNA primer to allow DNA polymerase a place to start primase

add info

primase

DNA directed RNA polymerase

okazaki fragments

short discontinuous sequences of DNA nucleotides that are synthesized during DNA replication, made of growing DNA strand and template strand

DNA ligase

joins fragments of DNA after DNA polymer 1 chops off RNA and adds new DNA

telomeres

at ends of chromosomes, nonsense DNA, no impact of loss, preserve information in genome

telomerase

enzyme active in sex cells, rebuilds telomeres for passing on info to offspring

exonuclease activity

enzymes that remove nucleotides from the ends of DNA, can remove in both directions

endonuclease activity

enzyme that cleaves a polynucleotide by separating nucleotides other than the two end ones

lead strand

continuous synthesis, 5’ —> 3’

lag strand

discontinuous synthesis, 3’ to 5’, gaps between rna and dna polymerase 3

experiment: 1 gene, one enzyme

scientists Beadle and Tatum, worked with fungus Neurospora, interested in amino acid synthesis (argenine), shoot x rays at fungi (causes mutations to DNA) 3 mutant strains that relate to argenine synthesis

one gene, one enzyme conclusion

a gene is the information on how to build a protein

experiment: breaking the code

Nirenberg, good at synthesizing DNA, works with bacteria (e coli)

1. lyses ecoli to put all cell contents into soln

2. synthesizes short stretches of RNA, add to cell lysis soln, measure what changes chemically

   1. added uracil produced phenylalanine, add in adenine produced argenine

breaking the code conclusion

the specific base sequence calls for specific amino acid

start codon

ATG/AUG codes for methionine

stop codon

3 types of sequences, do not code for specific amino acid, codes to stop

plus strand

strand of DNA in the correct order (5’ to 3’)

minus strand

reverse complement of plus strand, used as template by RNA polymerase

3 types of RNA polymerase in Eukaryotic transcription

RNA pol I, RNA pol II, RNA pol III

RNA pol I role

makes ribosomal rna (rRNA)

RNA pol II role

makes messenger rna (mRNA)

RNA pol III

makes transfer RNA (tRNA)

promoter region

region on DNA before the gene

transcription factor

binds to promoter region where it will be recognized and bound to by RNA polymerase

Termination (eukaryotes)

after we pass end of gene, endonuclease makes a cut to release RNA transcript

Termination (prokaryotes)

at the end of their genes there is a GC rich stretch followed by AT rich stretch, when GC region is synthesized forms a hair pin loop which snags on RNA pol, when AT region synthesized as RNA as AU, weaker than GC region, entire transcript lifts off

hair pin loop

loop formed in termination stage of prokaryotes when GC region is synthesized by RNA polymerase

rRNA transcriptional processing

uses RNA pol I, cuts and releases SSU and LSU and spaces from each other, spacers digested and removed, SSU and LSU fold up into their respective ribosome protein subunits

spacer regions

non critical sequence in ss RNA transcript for rRNA

SSU region

destined for incorporation into small subunits of ribosome

LSU region

destined for incorporation into large subunits of ribosome

mRNA transcriptional processing

RNa gets spliced to separate exons and introns, introns cut up and removed, exons reattached, add a 5’ cap, add a 3’ end poly A tail to protect 3’ end

introns

nonsense info

exons

actual instructions

5’ cap (7-methyl guanosine)

protects 5’ end of ENA from exonucleases (RNAses)

poly A tail

made by Poly A polymerase, protects 3’ end which slows the effect of RNAse attack, adds bunch of Adenines to the end that can be chopped off

trna transcriptional processing

rna transcript fold into 2* structure, expsoses anticodon, amino acyl tRNA synthetase attaches correct amino acid to 3’ end of folded RNA

anti codon

sequence of three nucleotides on tRNA molecule that corresponds with complementary code on mRNA

amino acyl tRNA synthetase

enzyme that attaches correct amino acid to 3’ end of folded RNA

translation goal

make a less specific protein based on mRNA instructions

E site

exit site on large sub unit (LSU) (on the left)

P site

petidyl site on LSU (middle)

A site

site on LSU where new amino acids enter (right side)

translation process

Initiation, Elongation, Termination

Initiation (eukaryotes)

SSU ribosome find mRNA, SSU binds to 5’ cap, initiator tRNA carrying methionine matches anti codon to start codon, large sub unit clamps down on SSU and tRNA sits in P site

Initiation (prokaryotes)

shine-dalgarno sequence in SSU rRNA, grabs onto mRNA, initiator tRNA carrying methionine matches anti codon to start codon, large sub unit clamps down on SSU and tRNA sits in P site

Elongation

new tRNA w matching anticodon enters A site w its amino acid, ribosome moves down mRNA (5’ to 3’) codon, amino acid (from p site) gets bound to amino acid from a site (covalent peptide bond), and gets released from its tRNA, tRNA with amino acid removed then exits via E site, then new tRNA w anticodon that matches codon exposed in the A site enters, process continues until stop codon reached

Termination process

release factor protein binds to stop codon, when ribosome shifts down to stop codon no tRNA or amino acid to join, bond holding growing peptide to tRNA in P site breaks

single base error/mutation from switching

base replaced by the wrong base in codon, codes for diff amino acid

insertion error/mutation

extra base added, changes order and codes for new amino acids