cmb405 - exam 2

three functions of genetic material

storage, replication, and expression

three alternate schemes of replication

semiconservative, conservative, and dispersive

semiconservative replication

* the actual scheme of of replication that DNA uses
* DNA is “unzipped” and each strand is replicated, leading to first generation progeny that have half new and half parental DNA

conservative replication

* original double stranded DNA is conserved and replicated
* first generation progeny have either 100% new or 100% parental DNA

dispersive replication

each individual strand in the replicated DNA has a random mixture of new and old strands

Meselson and Stahl

used density gradient centrifugation to test models of DNA replication by using heavy and light nitrogen to differentiate between parental and new strands of DNA

replication origin (*oriC*)

* specific site where replication begins
* prokaryotes have one and eukaryotes have multiple

replication forks

junctions where two strands of DNA separate to allow replication of each strand

helicase

enzyme that unwinds DNA

single-stranded binding proteins

keep DNA strands apart after they are unwound by helicase

gyrase

moves ahead of replication fork and relieves the stress caused by positive supercoiling, helping DNA unwind

DNA polymerase III

* enzyme that assists DNA replication by catalyzing the polymerization of dNTPs alongside a ssDNA strand
* requires a free 3’-OH and a template
* synthesizes in an antiparallel fashion (5’ → 3’)

primer

an oligonucleotide (DNA or RNA) can be added that “primes” replication via polymerase

leading strand

opens in the 3’ → 5’ direction and allows DNA polymerase to continuously synthesize DNA in the 5’ → 3’ direction

lagging strand

opens in the 5’ → 3’ direction and must be synthesized discontinuously via Okazaki fragments

primase

adds primers to the lagging strand

DNA polymerase I

removes RNA primers and replaces them with required nucleotides

DNA ligase

seals the gaps between Okazaki fragments

beta clamp

identifies where DNA polymerase III will bind next (at the end of RNA primer) on the lagging strand

beta clamp loader

holds beta clamp and loads it onto the next RNA primer

polymerase chain reaction (PCR)

* way to amplify a specific DNA sequence
* three steps: denaturing, annealing, and extension

95 degrees C

temperature for the denaturing step of PCR

50-60 degrees C

temperature for the annealing step of PCR

72 degrees C

temperature for the extension step of PCR

DNA sequencing reactions

just like the PCR reactions for replicating DNA but with the addition of ddNTPs

dideoxynucleotides (ddNTPs)

nucleotides with no 3’-OH group, making it impossible to elongate the strand of DNA

Sanger method

* ddNTPs in low concentration will terminate the chain every once in a while
* segments are sized via gel electrophoresis

telomeres

* found at the end of chromosomes
* more compact than the rest of DNA
* consist of a repeating sequence

telomerase

* reverse transcriptase
* uses RNA template to synthesize more DNA and extend telomeres

consensus sequence

a sequence that is the “average” of all sequences in those regions

TATA box

consensus sequence found in the promoter region of genes in archaea and eukaryotes

sigma factors

allows RNA polymerase to recognize the promoter region

\+1 start site

where the first nucleotide of the RNA strand is created in transcription

transcriptional bubble

formed where RNA polymerase interacts with DNA

RNA polymerase

uses a DNA strand as a template to synthesize RNA during transcription

TBP

TATA binding protein (transcription factor in eukaryotes)

TFIIB

will mark +1 start site (transcription factor in eukaryotes)

TFIIA

will help bend/melt DNA (transcription factor in eukaryotes)

TFIIH

helicase (transcription factor in eukaryotes)

terminator sequence in DNA

inverted repeats that RNA polymerase incorporates into the RNA strand

hairpin loop

* formed by transcription of the terminator sequence
* structure causes RNA polymerase to release the DNA transcript and fall off

information RNA

mRNA

functional RNA

tRNA, rRNA, snRNA

post-transcriptional modifications made to mRNA

5’ cap, poly A tail, splicing

5’ cap

* derivative of guanosine
* 5’-5’ linkage
* protects 5’ end from degradation
* shuttle mRNA out of the nucleus
* aids in translation

poly A tail addition

1. consensus sequence AAUAAA recognized near the 3’ end of RNA
2. triggers cleavage of mRNA transcript shortly after sequence (via nuclease)
3. poly A tail polymerase adds more A’s to the 3’ end of mRNA

functions of poly A tail

* protect 3’ end from exonucleases
* transport out of nucleus
* aids in translation
* confers stability (longer tail = longer half life)

splicing

pre-RNA is converted to mRNA by removing intron sequences

lariat

spliced out introns

spliceosome

a large RNA-protein complex that catalyzes the removal of introns from nuclear pre-mRNA

U1

recognizes 5’ splice site

U2

recognizes branch point during splicing of introns

nucleases

further degrade spliced out introns

group 2 introns

splice themselves out with no need for spliceosomes

carboxy terminal domain (CTD)

* extends off of RNA polymerase II
* can be phosphorylated
* phosphorylation can lead to recruitment of other complexes such as spliceosomes and capping enzymes
* RNA can be processed as it’s being made

amino acids

building blocks of proteins

amino group

found at the N-terminus of a protein

carboxy group

found at the C terminus of a protein

primary structure

sequence of amino acids

secondary structure

regular structure of a protein backbone as determined by interactions between molecules in the protein backbone

tertiary structure

overall 3D arrangement of a polypeptide in space

quaternary structure

multiple polypeptides interacting

“the genetic code contains punctuation”

the genetic code has start and stop codons

"“the genetic code is degenerate”

there are more codons than there are amino acids, so multiple codons can code for one amino acid

synonymous/silent mutation

a mutation that does not result in a change in the amino acid sequence

non-synonymous/missense mutation

a mutation that causes an amino acid substitution

nonsense mutation

a mutation that creates a premature termination codon

frameshift mutation

a mutation that alters the normal reading frame of mRNA

SNP (single nucleotide polymorphism)

a DNA sequence variation that occurs when a single nucleotide in the genome sequence is altered and the particular alteration is present in at least 1% of the population

ribosome

* uses RNA sequence to synthesize proteins
* composed of large and small subunits
* contain a mixture of protein and rRNA

tRNAs

* bring amino acids to their codon
* contain anticodons that bind to RNA codons in an antiparallel fashion

the wobble hypothesis

* base pairing isn’t so tight, and can sometimes tolerate mistakes
* nucleotide at 5’ end of tRNA anticodon is capable of pairing with more than one nucleotide at 3’ end of mRNA codon
* tRNA can recognize codons with variable third bases

aminoacyl tRNA synthetases

attach amino acids to specific tRNAs

A site

aminoacyl site where the charged tRNA is recruited

P site

ribosomal site of peptide linkage

E site

ribosomal exit site

Shine-Dalgarno sequence

* UAAGGAGGU
* consensus sequence in prokaryotes

initiation steps for protein synthesis in bacteria

1. initiation factor (IF) 1, 2, and 3 bind to the small subunit and help the ribosome bind to mRNA
2. first tRNA enters into the P site of the small subunit
3. large ribosomal subunit then binds to complete the APE sites

elongation steps for protein synthesis in bacteria

1. elongation factor (EF)-Tu recruits the next charged amino tRNA into the A site
2. peptide bond formation where the amino acid in the P site gets transferred to the amino acid in the A site
3. EF-G helps translocate the ribosome down three nucleotides
4. the empty tRNA in the E site leaves and a new charged tRNA enters into the A site

translation termination

release factor protein binds to the A site and releases the ribosome from mRNA

neomycin/gentamycin (aminoglycoside)

prevents ribosome assembly

tetracyclines

block the A site of the ribosome, prevents new tRNA from reading the codon

chloramphenicol

blocks peptidyl transfer from P to A tRNA (blocks peptide formation)

puromycin

binds in A site, blocks new tRNA from coming in

ricin

enzyme that cleaves part of the RNA molecule in the ribosome

housekeeping genes

genes that are consistently expressed across tissues, essential, carrying out cellular maintenance, and conserved across species

structural genes

encode proteins that are used in metabolism, biosynthesis, or play a structural role in the cell

regulatory genes

encode products (RNA or proteins) that interact with other sequences and affect transcription or translation

regulatory elements

DNA sequences that are not transcribed, but affect expression of other sequences

operon

a group of genes whose expression is controlled by a single promoter

lactose

* disaccharide composed of a glucose attached to a galactose
* binds to lac operon repressor and pulls it off of the operator, allowing RNA polymerase to transcribe genes Z, Y, and A

lac operon

* inducible operon
* controls the expression of proteins that utilize lactose for energy

LacZ

breaks lactose into galactose and glucose

LacY

* permease
* allows lactose to enter cells

operator

DNA element that binds repressor

repressor

binds to operator and prevents RNA polymerase from transcribing genes

catabolite activator protein (CAP)

* regulated by cAMP
* recruits RNA polymerase to the promoter in the lac operon

cAMP

* binds to and activates CAP
* when glucose levels are low, levels of this are high (and vice versa)

trp operon

* repressible operon
* controls expression of trp

trp

* corepressor
* binds to and activates the repressor in the trp operon