Week 2 Flashcards
chromosomes
carriers of genetic information
contain both proteins and nucleic acids
Griffith and Avery experiments
S bacteria: fatal to mouse, infectivity killed by heat
R bacteria: not fatal
smooth (S) bacteria contain something not destroyed by heat, can be passed on to R bacteria so that they kill and pass on
conclusion: molecule that carries heritable information is dna
Hershey and Chase
virus: consists of both dna and proteins
labeled dna with 32P, proteins with 35S
infected bacteria contain 32P but not 35S
Chargaff’s rules
dna, not proteins, is heritable material
A:T and G:C ratio always 1
A/T:G/C ratio variable from species to species
Franklin and Wilkens, Watson and Crick
showed dna is double helix (X-ray crystallography)
built model of structure of dna
nucleus of cell
the design and management center of the cell
stores dna
cytoplasm
production site of the cell, makes proteins
mRNA
messenger rna, used to transmit information from the nucleus to the cytoplasm
tRNA
transfer dna, adaptors from 4 base dna code to 20 amino acid protein code
ribosomes
make proteins using mRNA as template, amino acids as building blocks
large, complex molecules consisting of both proteins and rRNA
central dogma
Francis Crick, 1958
dna, rna, and proteins are linear, sequential polymers
each position in sequence is drawn from fixed alphabet (nucleotides for dna, rna—4, and amino acids for proteins—20)
flow of primary sequence information: conversion between alphabets (translation)
no way to convert protein alphabet back to nucleic acid
transcription from dna to rna, then translation from rna to protein
gene expression
regulation of this determines if cell is liver, muscle, nerve
levels vary from cell to cell in same organism, at times in development, and with outside signals
RNA polymerases
enzymes that carry out transcription
synthesis of rna from 5’ to 3’
signals on dna that tell rna polymerases where to start/stop (subject to regulation)
promoter
defines where transcription should begin
rna poly. binding site
then coding region
prokaryotes
have no nucleus
single cellular (but may join together)
can live in diverse temps
can grow and evolve quickly
eukaryotes
have nucleus
are multicellular
have other intracellular organelles (some of which are thought to have evolved from invading bacteria—mitochondria, chloroplasts)
prokaryotic genes
minimal gene
promoter—binding site for rna polymerase
ribosome binding site (initiate protein synthesis in mRNA)
coding sequence—encodes protein synthesis
transcriptional terminator—stop mRNA synthesis
often multiple protein coding regions controlled by single promoter (operon)
-polycistronic: more than one protein encoded in single mRNA molecule
polycistronic mRNA
more than one protein encoded on single mRNA molecule
common in prokaryotes
eukaryotic genes
5’ caps and 3’ poly-A- tails
require general transcription factors
most have introns (noncoding regions) that are spliced
can be simple of very large
introns and exons
noncoding and coding regions in the dna
splicing
requires intronic signal sequences
lariat formed from introns
alternative splicing
can be differentially regulated
enables a mRNA to direct synthesis of different protein variants
translation
once mRNA is out of nucleus, it is translated into proteins—different alphabet
genetic code
genetic code
directional: 5’ to 3’ mRNA
no doves to indicate start or stop of words
6 different reading frames from dna to proteins
Kozak sequence
all proteins start with amino acid methionine
A/GccAUGG/A
stop signal
TGA, TAA, TAC (UGA, UAA, UAC)
tRNA synthetases
charge tRNA with amino acids
tRNA anticodons
base pair with the correct codon on mRNA (after tRNA synthetases charge)
rRNA
ribosomal rna, most abundant rna in most cells
form the core of the ribosome and catalyze protein synthesis
ribosome sites for tRNA
A—admission of codon if mRNA, checking and decoding
P—peptide synthesis, consolidation, elongation, transfer of peptide chain to site A
E—preparation of now uncharged tRNA for exit
electrophoresis
separation of charged biomolecules in a gel matrix in which an electric field has been established
separates proteins, rna, dna
phosphate moieties of dna/rna
provide nucleic acid molecules with constant, negative charge to mass ratio
charge of protein depends on primary structure and ph of the solution
equipment for gel electrophoresis
power source, electrophoresis tank, anode (+) and cathode cable, support, comb
bands on electrophoresis gel
population of identical molecules that are not exactly identical in migration behavior -> distribution
variables affecting: molecular, thermal, gel heterogeneity, observation error
the southern blot
dna separated on gel and dna as the probe
the northern blot
rna separated on gel and dna as the probe
charge/mass ratio
constant for dna and rna (so just separate by size)
unique for every protein
two solutions for unique charge:mass ratio in proteins
treat proteins to give them uniform charge
take advantage of this property to separate proteins
SDS (sodium dodecyl sulfate)
strongly denaturing detergent (disrupts 2o-4o structures)
binds and confers negative charge to proteins (charge ~ mass)
denaturing protein gel electrophoresis
proteins unfolded by loss of disulfide and hydrogen bonds
SDS coating -> uniform charge/mass ratio
mobility is inverse of mass
proteins of known mass used as standards to calibrate gel
isoelectric focusing
not denatured, ph gradient within gel
protein migrated from well to ph where is has no net charge (isoelectric pt, pi)
2d gel
electrophoresis in 1st rim by isoelectric point (native)
electrophoresis in 2nd din by size (reduced/SDS)
antibodies
proteins that bind strongly with specific 3D structure (specific protein)
produced naturally by immune system to help in detection of foreign antigens
used to identify proteins in bio samples
Western blot
detection of specific protein structure using antibody
