bio exam re2

central dogma (theory)

genetic information flows in one direction

• from DNA to RNA to Protein

RNA replication

RNA is copied without a DNA intermediate

• used by viruses (to make more viruses)

reverse transcription

synthesis of DNA using an RNA template

• used by viruses

• eg. HIV (retrovirus infection)

what process is being used by the virus

RNA replication

what process is being utilized by the parasite

DNA replication

2 polymers that make up a chromosome

DNA & proteins

frederick griffith experiment

DNA is genetic material

• transformation of mice

transformation

process by which an organism acquires exogenous DNA

proteins but not DNA are

killed with heat

• separates DNA but wont kill them

• eg. Fever: degrades proteins

• eg. DNA extraction & PCR: kills DNAases/proteases; separates DNA strands

avery-macleod-mccarty experiment

confirms griffith’s conclusion that DNA is the “transforming” particle

hershey and chase

DNA is genetic material

• experiment with bacteriophages + radioactivity

5 steps of infection

1. virus attaches to bacterial wall

2. virus injects genetic material into cell

3. viral DNA disrupts bacterial DNA + uses cellular machinery to make viral components (proteins + more DNA)

4. new viruses assemble

5. New viruses are released to infect other cells and restart the cycle

nucleotide sequences

carry information to make primary protein structures

transcription

process of making RNA (from DNA)

• DNA is template

• coding / non-coding RNA

coding RNA

encodes for proteins

• eg. mRNA

non-coding RNA

“housekeeping” RNA that does not encode for proteins

• eg. tRNA, snRNA, rRNA, miRNA

RNA polymerase

multi-unit enzyme; synthesizes RNA from DNA template

transcription in eukaryotic vs prokaryotic

spatially and temporarily separated in eukaryotic but simultaneous in prokaryotic

RNA polymerases (bacteria)

1 type

• 4 catalytic subunits +1 regulatory subunit

RNA polymerases (eukaryotes)

3 types

• RNA polymerase I - rRNA

• RNA polymerase II - mRNA/snRNA

• RNA polymerase III - tRNA

transcription 1st step: initiation

RNA polymerase binds, DNA unwinds, and transcription bubble is created

transcription 2nd step: elongation

RNA synthesized from 5’ to 3’ using ribonucleotides by RNA polymerase

transcription 3rd step: termination

transcription stops, RNA-DNA hybrid and bubble dissociates, RNA polymerase is released, DNA rewinds

the structure of a gene determines

the START and STOP of transcription by RNA polymerase

monocistronic

gene under 1 promoter codes for 1 protein

polycistronic

multiple genes under 1 promoter codes for multiple proteins

promoter forms a

recognition and binding site for RNA polymerase

promoter

region of DNA upstream of a gene where proteins bind to initiate transcription

• eg. prokaryotic promoter

transcription factors

proteins that to the -35 and -10 elements and recruit RNA polymerase

enhancer

regulatory DNA sequences; enhance transcription of a gene when bound by transcription factors

• eg. eukaryotic promoter

ribonucleotides consist of

A, U, C, and G

introns

non-coding regions of DNA

exons

coding regions of DNA

splicing

introns are removed so protein synthesis can occur

• occurs in nucleus

pre-mRNA

synthesized by RNA polymerase during transcription

• contains introns + exons

mRNA

product after splicing occurs (only exons)

complex that helps remove introns

spliceosome complex

why do eukaryotes have introns

• help regulate gene expression

  • splicing process needed for extracellular export of mRNA

  • introns contain sequences that control gene activity

• alternative splicing: a single gene codes for more than one polypeptide by changing how a gene is spliced

  • results in different proteins being made

  • associated w/ ~15% of human genetic disorders

mRNA capping

adding a 5’ cap on mRNA

5’ cap

specially altered nucleotide at the 5’ end that:

• regulates nuclear export, prevents degradation by exonucleases, promotes translation, promotes 5’ intron excision

polyadenylation

adding a poly-A tail to mRNA

poly-A tail (3’)

long chain of adenine nucleotides added to mRNA to increase stability and prevent degradation

transcription regulation: activators + repressors

promote transcription; prevent transcription

operators

DNA sequence near the promoter

• where activators/repressors bind

housekeeping (constitutive) genes

genes essential for cell survival and expressed all the time

translation

making proteins from messenger RNA (mRNA)

ribosome

2 subunits that build peptides from mRNA

transfer RNA (tRNA)

interprets mRNA and brings amino acids to ribosome (anticodon)

• 1 tRNA for each amino acid

codon

mRNA sequence; 3 sequential nucleotides that dictate an amino acid that tRNA brings to the ribosome

prokaryotic vs eukaryotic ribosome subunits

pro: 70S ribosome

euk: 80s ribosome

how many amino acids does this mRNA encode for?
GCT CTA TGG GGA CCT

5

what bond connects amino acids

peptide bonds

code bias

different species may prefer different codons

exception

UGA is universally a STOP codon

• exception: codes for trp in vertebrate mitochondria

wobble hypothesis

explains why multiple codons code for the same amino acids

F. Crick & S. Brenner experiment

• how order of nucleotides encodes for the order of amino acids using mathematical modeling

• how single nucleotide insertions/deletions change proteins (point mutation)

• confirmed that genetic code was triplet code

reading frame

codons, every 3 nucleotides

2 functions of ribosome during translation

1. decode mRNA

   1. recruit proper tRNA

2. form peptide bonds

   1. peptidyl transferase: forms peptide bonds between amino acids

shine-dalgarno sequence (prokaryotic)

ribosomal binding site in bacterial/archaeal mRNA

kozak consensus sequence (eukaryotic)

nucleic acid that functions as protein translation initiation site

initiation factor

brings ribosomal components to mRNA

A site of ribosome (aminoacyl)

first binding site in the ribosome

• exception: initiator tRNA

P site of ribosome (peptidyl)

holds tRNA as polypeptide chain grows

E site of ribosome (exit)

uncharged tRNAs exit the ribosome

elongation factors

bring charged tRNAs to the ribosome and translocate tRNAs inside the ribosome

ribosomal process

1. subsequent RNAs bind to A-site

2. after peptide synthesis, tRNAs exit via the E-site

3. termination: completed polypeptide is released and ribosome dissociates

stop codon

tells ribosome to stop translating

• no acetyl-tRNA enters A-site

• release factors

release factors

enter and cleave bond between peptide and tRNA

3’ UTR (untranslated region)

aids in termination

• folds into stem-loop; helps disassociate the ribosome from transcript once stop codon is reached

image

prokaryotic translation

• shine-dalgarno sequence (AGGAGGU))

• ribosome-binding site (RBS) present

prokaryotic or eukaryotic translation

• coupled transcription + translation

  • transcribing DNA + ribosome translating mRNA

• ribosome-bonding site (RBS) feature of prokaryotic transcripts

conservative model

parental DNA remains together while newly formed daughter strands become paired

semi-conservative

each of the 2 parental DNA strands acts as a template for new DNA to be synthesized so that each newly formed daughter strands is paired with an “old” strand

dispersive model

both copies of DNA have double-stranded segments of parental DNA and newly formed DNA interspersed

meselson-stahl experiment

The experiment proved that DNA replication is semiconservative: every time a cell divides, each of the two resulting DNA molecules consists of one strand from the original molecule and one newly synthesized strand

polymerase chain reaction (PCR)

allows scientists to rapidly replicate lots of DNA

gel electrophoresis

allows scientists to determine what DNA was replicated in PCR

• where smaller DNA travels further

end-replication problem in eukaryotes

loss of genetic information due to RNA primers inability to “clip onto” the lagging strand (lack of space)

telomeres

protective, repetitive, non-coding regions at the end of linear chromosomes; prevents loss of coding regions of DNA during replication and unraveling/sticking of chromosome ends

telomerase

protein found in eukaryotic cells that extend telomeres by writing telomeric DNA

• in cancer, egg, sperm, and stem cells

centrifugation

separates heavy from light DNA

DNA polymerase

“proofreads” the replicated DNA for mitosis

• replaces errors

exonuclease activity (nuclease)

removal and replacement of incorrect nucleotides by cleaving the phosphodiester backbone

DNA ligase

seals gaps in DNA backbone

mismatch repair

incorrectly added bases are detected, removed, and replaced

base excision repair

only the damaged base is removed and replaced

nucleotide excision repair

1+ damaged bases are removed and replaced

if errors in replication are not repaired

mutations occur

induced mutation

caused by environment

• chemicals, xrays, UV light, lab experiments, etc

spontaneous mutation

naturally caused, randomly occurring

DNA mutations in non-coding DNA

most mutations occur in this type since it makes 97% of our DNA

• affects the expression of genes

• mutations still occur in coding regions

codon

sequence of 3 nucleotides

• represents an amino acid or start/stop signal of protein synthesis

point mutation

change of a single nucleotide for another

missense (point) mutation

one nucleotide change resulting in a different amino acid

• sickle cell anemia

nonsense (point) mutation

one nucleotide change resulting in a stop codon (UAA, UGA, UAG)

• duchenne muscular dystrophy

silent (point) mutation

single nucleotide change resulting in no change of amino acid

frame shift mutation

addition/deletion of nucleotide(s) that shift all following amino acids making them wrong (frameshift error)

• creates new proteins useless to the cell (stop codons)

• more severe than point mutations

• thalassemia + tay sachs disease

insertion/deletion of whole codons

no frameshift errors; may pose severe effects

spontaneous mutations

result from intracellular processes

• meiosis, inherent instability of nucleotide bases, DNA polymerase error, base pair degradation

induced mutations

result from external mutagens