Unit 3 Flashcards
Friedrich Miescher
investigated chemical composition of DNA using pus cells
discovered DNA but called it nuclein
found it to be slightly acidic and composed of large amounts of phosphorus and nitrogen
caused debate of if protein or nuclein is hereditary material
Hammerling experiment
removed caps of algae and they regrew, removed feet of algae (included nucleus) and they did not regrow
concluded hereditary info is found in the nucleus
Griffith
discovered transforming principle in experiments with lethal/non-lethal strains of pneumonia
Hershey & Chase
confirmation of DNA
infected bacteria with 2 different kinds of radioactive viruses (S and P)
Chargaff’s rule
In DNA, percent composition of adenine is the same as thymine, and percent composition of cytosine is the same as guanine
DNA structure
a polymer of nucleotides
each of the four types of nucleotides contain a phosphate group, a deoxyribose sugar, and one of four possible nitrogen-containinng bases (adenine, guanine, cytosine, and thymine)
Linus Pauling
discovered many proteins have helix-shaped structures
Rosalind Franklin
used X-ray diffraction to analyze structure, patterns in pictures suggested a double helix shape
based on reactions with water, concluded that nitrogenous bases were located on the inside of the helix and sugar-phosphate backbone was located on the outside
Watson and Crick
deduced structure of DNA using other’s works
what is DNA
deoxyribonucleic acid
what chromosomes and genes are made of
made up of repeating nucleotide subunits
types of nitrogenous bases
adenine and guanine are purines (two rings of nitrogen atoms)
cytosine and thymine are pyrimidines (one ring of nitrogen atoms)
bonds in nucleotides
nitrogenous base is attached to the 1’C of the sugar by a GLYCOSYL bond
phosphate group is attached to a 5’C by a PHOSPHODIESTER BOND
DNA consists of 2 antiparallel strands of nucleotides. what does this mean
parallel, but running in opposite directions; the 5’end of one strand of DNA aligns with the 3’ end of the other strand in a double helix
hydrogen bonds in DNA
2 hydrogen bonds between A and T
3 hydrogen bonds between G and C
when does DNA replication occur
S stage of interphase
semi-conservative model
two parent strands are separated and a new complementary replacement strand is built for each
new DNA molecules would consist of one parent strand and one new strand
meselson & stahl experiment
concluded DNA replication is semi-conservative
used nitrogen isotopes and bacteria
three basic phases of DNA replication
initiation - portion of DNA is unzipped to exposes bases for new base pairing
elongation - two new strands of DNA are assembled using parent DNA as a template and then re-formed into double helices
termination - replication is done, two new DNA molecules separate, replication machinery is dismantled
when does replication begin
proteins bind at the replication origin
difference in replication origin in prokaryotes and eukaryotes
prokaryotes - one replication origin
eukaryotes - several
enzymes for strand separation
DNA helicase - the enzyme that unwinds double-helical DNA by disrupting the H bonds between the nitrogenous base pairs
Single-stranded binding proteins (SSBs) - a protein that prevents exposed strands from re-attaching together by blocking hydrogen bonding
topoisomerase (gyrase) - relieves the stress and kinks in the strands caused by separated by breaking and resealing the DNA strand
a new DNA strand is only made in the ________ direction
5’ to 3’
leading strand vs lagging strand
leading strand - copies continuously towards the replication fork
lagging strand - copies discontinuously, opposite the replication fork
RNA primase and RNA primers
RNA primase enzymes begin the replication process by building a small complementary RNA segment (RNA primer) on the strand at the beginning of the replication fork
RNA primer serves as a starting point for replication
RNA primer only needs to be added once for the leading strand
DNA polymerase III
attaches to 3’ end of each primer and begins assembling new DNA strands
okazaki fragments
series of short segments synthesized in the 5’ to 3’ direction copied discontinuously
each fragment is initiated by an RNA primer
fragment will eventually run into RNA primer
DNA polymerase I
removes RNA primers from both leading and lagging strands, appropriate DNA nucleotides replace them
DNA ligase
links together Okazaki fragments through creation of phosphodiester bonds
errors in DNA replication
mispairing nucleotide bases
strand slippage that causes additions or omissions of nucleotides
correcting errors
DNA polymerase II - proofreads newly synthesized DNA. incorrect base is removed and replaced
telomeres and telomerase
telomeres - repeating, non-coding sequences at the end of chromosomes, protective cap
telomerase - extends telomeres, can add DNA bases at 5’ end
difference between RNA and DNA
AUCG
ribose sugar
single stranded
how is RNA involved in protein synthesis
mRNA - template for translations
tRNA and rRNA - involved in translation of mRNA
Archibald Garrod
studies on alcaptonuria showed that having the black urine phenotype was due to what Mendel called a recessive inheritance factor. having this defective factor resulted in the production of a defective enzyme
Beadle and Tatum
bread mold experiments showed that a single gene produces one enzyme
Jacob and Monod
hypothesized existence of messenger RNA (mRNA)
mRNA
RNA that contains the genetic info of a gene and carries it to the protein synthesis machinery
provides the info that determines the amino acid sequence of a protein
base sequence is complementary to gene DNA sequence
Jacob, Brenner, Meselson
confirmed the messenger RNA hypothesis with experiments with bacteria and viruses
when bacteria were infected by a virus, virus-specific RNA molecule was synthesized and became associated with bacterial ribosomes. RNA molecule had complementary base to DNA and carried genetic info to make viral protein. viral RNA molecule was synthesized and was not a permanent part of the bacterial ribosomes
genetic code
a set of rules for determining how genetic info in the form of a nucleotide sequence is converted to an amino acid sequence of a protein
triplet hypothesis
a proposal that the genetic code is read three nucleotide bases at a time
each triplet is called a codon
genetic code is always interpreted in terms of the ______ codon rather than the nucleotide sequence of the _______
mRNA, DNA
three important characteristics of the genetic code
genetic code is redundant - more than one codon can code for the same amino acid
3 codons that do not code for amino acids but for stop signals to end protein synthesis
genetic code is continuous - reads as a series of 3-letter codons without spaces, punctuation or overlap
shift of one or two nucleotides in either direction can alter codon groupings and result in an incorrect amino acid sequence
genetic code is nearly universal - almost all organisms build proteins with this genetic code
important implications for genetic techniques, such as cloning
the central dogma of genetics describes the
transfer of genetic info from DNA, to RNA, and finally to proteins
gene expression
synthesis of a protein based on the DNA sequence of a gene
steps in gene expression
transcription - mRNA is synthesized based on the DNA template of a gene
translation - protein is synthesized with an amino acid sequence that is based on the nucleotide sequence of the mRNA
antisense vs sense strand
template/transcribed DNA strand vs coding/untranscribed DNA strand
three phases of transcription
initiation - transcriptional machinery is assembled on the sense strand. RNA polymerase binds to the promoter region of the sense strand, DNA is unwound
elongation - RNA polymerase synthesizes a strand of mRNA that is complementary to the sense strand of DNA
termination - RNA polymerase detaches from the DNA strand when it reaches a stop signal. the mRNA strand is released and the DNA double helix reforms
mRNA modifications
modifications covert precursor mRNA to mature mRNA before it is transported across the nuclear membrane in the cytoplasm for protection
done through capping and tailing, and splicing
capping
5’ cap consisting of 7-methyl guanosine units is added
protects the mRNA from digestion from nucleases and phophatases
tailing
a poly-A tail is added to the 3’ end
consists of approximately 200-300 adenine ribonucleotides added by poly-A polymerase
protection from degradation, facilitates attachment to ribosomal complex
exons, introns, spliceosomes, SnRNPS
exons - coding regions
introns - non-coding regions interspersed on exons
spliceosomes - cut out introns and rejoin exons
small nuclear ribonucleoproteins - play a key role in RNA splicing
true or false: prokaryotic DNA does not contain any introns
TRUE
removal of non-coding regions
mRNA splicing removes introns from pre-mRNA and joins exons together
snRNPS recognize and bind to regions where exons and introns meet
major components of translation
mRNA
tRNA
ribosomes
translation factors
tRNA
each tRNA has 2 functional regions:
anticodon loop - sequence of 3 nucleotides that is complementary to a specific mRNA codon
acceptor stem - at 3’ single-stranded region where amino acid is attached
tRNA links condons on mRNA to corresponding amino acid for protein synthesis
aminoacyl-tRNA synthetase - enzyme responsible for attaching amino acid to tRNA
ribosomes in translation
- cell structure in cytoplasm composed of proteins and ribsomal RNAs (rRNAs)
- site of protein synthesis
- each ribosome has a large and small sub-unit composed of different proteins and rRNA molecules
- has a binding site for mRNA and 3 binding sites for tRNA
initiation in translation
initiation - proteins called initiation factors assemble the small ribosomal sub-unit, mRNA, initiator tRNA and large ribosomal sub-unit for start of protein synthesis
mRNA is sandwiched between the two ribosome subunits
ribosome reads downstream until it reaches start codon - AUG. the corresponding tRNA carrying methionine binds to the P (peptide site)
three binding sites for tRNA
P (peptide) site - contains tRNA with growing polypeptide attached to it; at initiation, initiator tRNA carrying methionine binds to the P site
A (amino acid) site - contains tRNA with next amino acid to be added to polypeptide chain
E (exit) site - uncharged tRNA that has lost amino acid is ejected at the E site
elongation in translation
cycle of 4 steps is rapidly repeated. tRNA with attached polypeptide is in P site, tRNA carrying next amino acid enters A site. polypeptide chain is transferred to amino acid of tRNA in A site which makes the chain one amino acid longer. mRNA moves forward by one codon and tRNA with polypeptide is now at P site. empty tRNA exits from E site
termination in translation
termination begins when a stop codon on mRNA is reached.
a protein called a release factor cleaves polypeptide from last tRNA
ribosome splits into its subunits
ribosome and mRNA are recycled
mutation
permanent change in nucleotide sequence of a cell’s DNA
can arise spontaneously (DNA replication error) or be induced by a mutagen (ex. chemicals and radiation)
point mutation, types
single base change
silent mutation - no amino acid change, redundancy in code
missense - change amino acid
nonsense - premature stop codon
frameshift mutation
shift in the reading frame, changes everything “downstream”
insertion or deletion of bases
chrosome mutations
involves large segments of DNA (chromosomes)
deletion
duplication
inversions
translocations
why regulate genes
maintain homeostasis
multicellular organisms have specialized cells
gene regulation
blocks transcription of genes to save energy by not wasting it on unnecessary protein synthesis
regulation of gene expression in prokaryotes
in prokaryotes, many genes are clustered together in a region under the control of a single promoter - region is called an operon
all genes in the operon are transcribed together into one continuous mRNA strand - polycistronic mRNA
individual proteins are then synthesized from mRNA
operons
found in prokaryotic genomes
group of genes that are transcribed together
consists of a coding region and a regulatory region
parts of an operon
Promoter - sequence of DNA where RNA polymerase binds and begins transcription
Repressor - proteins that binds to an operator site, preventing transcription of genes in operon
Operator - regulatory DNA sequence to which a repressor protein binds - inhibits transcription initiation
Genes
repressible operons vs inducible operons
repressible operons are on and can be turned off ex. trp operon
inducible operons are off and can be turned on ex. lac operon
gene regulation for eukaryotes
methods for regulating eukaryotic gene expression are complex and require large number of steps
do not use operon systems
control mechanisms fall into four general categories:
- transcriptional (as mRNA is being synthesized)
- post-transcriptional (as mRNA is being processed)
- translational (as protein is being synthesized)
- post-translational (after protein has been synthesized)
transcriptional control
each gene has its own promoter
transcriptional control regulates which genes are transcribed and/or rate of transcription
access to promoters is provided by loosening DNA molecules from histones
post-transcriptional control
controls availability of mRNA molecules to ribosomes
masking proteins bind to mRNA and inhibit further processing
translational control
controls how often and how rapidly mRNA transcripts will be translated into proteins
variation of the length of the poly (A) tail on mRNA is related to rate of translation
initiation of translation stage can be blocked
post-translational control
controls when proteins become fully functional, how long they are functional, and when they are degraded
ubiquitin-tagged proteins are degraded (DEATH TAG)
recombinant DNA
cutting DNA fragments from different sources and recombining them together
purpose - to investigate genetic disorders, production of drugs (ex. insulin)
restriction endonucleases / restriction enzymes
- molecular scissors
- recognize a specific DNA sequence and cuts the strands at a particular position or “recognition site”
- isolated and purified only from bacteria
how do restriction enzymes work
- scans DNA and binds to its specific recognition sequence.
- disrupts the phosphodiester and hydrogen bonds
- results in 2 DNA fragments
different DNA fragment ends produced after digestion by different restriction enzymes
sticky ends: DNA fragment ends with short single-stranded overhangs
blunt ends: DNA fragment ends are fully base paired
restriction endonucleases: recognition sites
each restriction endonuclease recognizes its own specific recognition site (specific DNA sequence)
usually 4-8 base pairs long, characterized by a complementary palindromic sequence
DNA ligase role with restriction endonucleases
DNA ligase rejoins cut strands of DNA together by reforming a phosphodiester bond
DNA ligase joins sticky ends and also blunt ends
gel electrophoresis
technique used to separate charged molecules based on their size
restriction enzymes digest DNA into smaller fragments of diff lengths; diff DNA samples are loaded into wells of gel; negatively charged electrode at the end where wells are located with positively charged electrode at opposite end. negatively charged DNA migrate towards positive end due to attraction; smaller DNA fragments migrate faster
plasmids
small, circular double-stranded DNA that can enter and exit bacterial cells
lack a protein coat
independent of bacterial chromosome
can be used to introduce foreign DNA into the bacteria to have the desired gene transcribed and translated
plasmid mapping & restriction maps
diagrams that show restriction enzyme recognition sites and distances, measured in base pairs between the sites
allows scientists to determine which plasmids might be most suitable for a particular recombinant DNA procedure
used to determine which restriction enzyme should be used to cut the plasmid
genetic engineering pioneers
Stanley Cohen (plasmids) and Herbert Boyer (restriction enzymes) experiments for selecting, recombining and transforming new genes into bacteria ex. producing hormones like somatotropin and insulin with genetic engineering
polymerase chain reaction
small sample of DNA can be amplified to make multiple copies of a desired DNA fragment, exponential growth using repeated cycles
one cycle:
- double stranded DNA is denatured with heat to separate strands by breaking H bonds (no DNA helicase or gyrase)
- DNA primers anneal to complementary template DNA that bracket the desired DNA sequence
- Taq polymerase add complementary nucleotides to synthesize new DNA strand
repeat cycle
restriction fragment length polymorphism (RFLP)
polymorphism - any difference in DNA sequence (coding or non-coding) that can be detected between individuals
restriction fragment length polymorphism analysis - technique that compares different lengths of DNA fragments produced by restriction endonucleases to determine genetic differences between individuals by using complementary radioactive probes
RFLP analysis
- digest DNA using restriction enzymes
- run digested DNA on gel using gel electrophoresis
- expose gel to a chemical to denature double-stranded DNA to become single-stranded
- southern blotting
DNA sequencing
determine sequence of base pairs for genes
sanger dideoxy method
sanger dideoxy method
dideoxy nucleotides lack an OH group
reaction stops when a dideoxynucletoide becomes incorporated (DNA polymerase cannot add next complementary base)
chain termination results in different DNA fragment lengths
separate different DNA lengths by gel electrophoresis, sequence can be read from gel in ascending order
