Genetics Exam II

Normal form of DNA name

B form (right handed helix, 10 bp per turn), wet DNA

Dried out form of DNA

A form (twists tighter, still right handed), 10.9 bp per turn

DNA form twisted the wrong way

Z form (left hand helix, 12 bp per turn), supercoiling, transcriptional regulation, 12 bp per turn

plasmids

small accessory units of DNA, contain maybe one gene, exchanged between prokaryotes, used in horizontal gene transfer

NAPs

nucleoid associated proteins

• in prokaryotes, anchor the DNA in loops

euchromatin

“true” chromatin, decondenses during cell cycle, available for transcription, get read and expressed

heterochromatin

condensed throughout cell cycle, not transcribed

constitutive chromatin

type of heterochromatin, like centromeres or telomeres, always wound up because we don’t need to read it

facultative chromatin

type of heterochromatin, like Barr Bodies or DNA for lung cells in skin cells

useful, but not expressed in a particular cell for some reason (can be undone during replication)

describe the histone structure

4 pairs of histones (H2A, H2B, H3, H4) that form an octomer, and histone H1 which is longer and anchors DNA on the nucelosome.

tails are rich in arginine and lysine, which are positive and attract the negative DNA backbone

Nucleosome

complex of DNA with a histone

Acetylation

HATs add an acetyl group to a histone tail, neutralizes the positive charge so the histones relax the hold on the DNA, enhances transcription

HATs

histone acetyl transferases, perform histone acetylation

histone deacetylation

HDs take away added acetyl group, histones regain positive charge and grab more tightly to DNA again, winding chromatin tighter

HDs

histone deacetylases, perform histone deacetylation

histone methylation

by adding methyl groups to charged polar histones, they interact more poorly with water and group together, holding the chromatin more tightly

centromeres

constitutive heterochromatin region on chromosome made of repetitive DNA

• joins sister chromatids

• forms the kinetochore where spindle microtubules attach

• contains histone CENP-A (centromere protein A)

telocentric

centromere is at the tip

acrocentric

centromere is right below the tip

submetacentric

centromere is almost at the middle

metacentric

centromere is at the middle of the chromosome

p arm

shorter arm of chromosome on one side of the centromere

q arm

longer arm of chromosome on one side of the centromere

telomeres

repetitive constitutive heterochromatin

• prevent DNA from sticking together

• we can lose it during replication

• protects the ends

• can repair with telomerase

• contain a 3’ overhang

telomerase

enzyme that replicates telomeres, especially when making gametes (gametogenesis)

at what distance are two genes unlinked

50 cM

cM

centimorgan, refers to 1 percent chance the two will recombine compared to each other

cis configuration

for a heterogametic chromosome pair (both dominant and recessive alleles), the dominant alleles for two genes are on the same chromosome, will most likely see AB and ab gametes (coupling)

trans configuration

both dominant and recessive alleles on one homologous chromosome, more likely to see Ab or aB gametes (repulsion)

how to determine the order of genes

for a three point test cross, it is the one that flips in a double recombinant

how to tell if we have coupling/repulsion

for coupling (cis transfiguration) we’ll see more of the AB and ab types, less of the heterozygous types

replicon

group of DNA replicated from one origin of replication (prokaryotes have one replicon, just one for both strands)

replication fork

where the DNA is unwound to replicate

what direction is DNA synthesized

5’ to 3’ of the daughter strand, read in the 3’ to 5’ direction by DNA polymerase

continuous synthesis

happens on the leading strand, one RNA primer and nonstop replication

discontinuous synthesis

happens on the lagging strand, multiple RNA primers, forms okazaki fragments

DNA replication initiation in prokaryotes

• OriC sequence recruits initiator proteins (DnaA)

• DnaA boxes bind a single DnaA, we have a bunch of these in the OriC sequence

• this puts strain on the DNA, have strand separation at AT rich DNA unwinding elements (DUEs)

• AT pairs are easier to separate because they only have 2 H bonds

• access for helicase, forms replication bubble

unwinding of DNA in prokaryotes

• helicase breaks H bonds between base pairs of DNA and separates the strands

• moves in the 5’ to 3’ direction on the lagging parental strand

• single strand binding proteins (SSBPs)

• bind to the DNA after it’s separated to prevent reannealing

• gyrase (Type II topoisomerase) relieves strain farther down an unwound double helix, cuts both strands and allows it to untwist, prevents supercoiling (comes in after the bubble has formed)

DNA synthesis in prokaryotes

• primase lands on the DNA and creates an RNA primer

• DNA polymerase III adds DNA to 3’ end of primer and then the existing DNA, proofreads in the 3’ to 5’ direction, has an exonuclease component that can take out and replace errors as necessary

• DNA polymerase I removes RNA primer, 5’ to 3’ exonuclease removes one nucleotide then the polymerase adds back the correct nucleotide at the same time

• ligase makes phosphodiester bonds joining DNA fragments on lagging strand

Termination of replication in prokaryotes

terminator sequence (Ter) in prokaryotes

binds terminator protein (Tus), preventing unwinding

one fork stalls at the terminator, eventually the other fork will meet it there

mismatch repair

enzymes that proofread DNA post replication, take out errors and fix them

DNA replication initiation in eukaryotes

• multiple origins of replication, all with different sequences

• recruit Origin Recognition Complexes (ORCs) of proteins, which load during G1 phase

• ORC anchors helicase (made of MCM2-7 proteins)

• helicase starts during S phase

• licensing factors determine which origins are active

unwinding DNA in eukaryotes

• helicase is different than prokaryotes, made of a donut of MCM2-7 proteins, move in opposite directions, built during G1 phase before the bubble forms

• class I topoisomerase cuts and unwinds DNA to prevent supercoiling, usually cuts one strand

• naturally takes out histones and unwinds nucleosomes, they form again after

DNA synthesis in eukaryotes

• DNA polymerase alpha has primase activity, starts synthesis, makes the RNA primer and adds DNA to the 3’ end

• DNA polymerase epsilon completes leading strand synthesis (elongation)

• DNA polymerase delta joins the complex for lagging strand synthesis, with its own helicase, 3’ to 5’ error correction

• RNA primers are removed by FEN1, a 5’ to 3’ exonuclease, the next fragment will fill in the gaps

complete linkage

• crossing over never happens between 2 genes

• 0% chance of recombination

• very close together

incomplete linkage

• crossing over happens sometimes, not always between genes

• <50% chance of recombination

• <50 cM apart

unlinked on the same chromosome

• crossing over always happens

• 50% chance of recombination

• >50 cM apart

unlinked on different chromosomes

• 50% chance recombination

Genetic map

• maps that show where on a chromosome a gene is, in units of cM in linkage groups

physical map

units of nucleotide base pairs in chromosomes, showing where genes are

interference

recombination at one site affects recombination at another

calculate CoC

observed double recombinants/expected

expected = RF1*RF2×1000

calculate interference

1-CoC

mRNA

messenger RNA, protein coding information for ribosomes

tRNA

transfer RNA, binds mRNA, delivers amino acids

rRNA

ribosomal RNA, structural component of ribosomes, helps RNA binding proteins in the small subunit

snRNA

small nuclear RNA, part of spliceosome

miRNA

micro RNA, regulates mRNA utilization

RNA polymerase in pro. and euk.

• single enzyme complex with accessory proteins

• opens double helix locally

• prokaryotes have one, sigma factors allow binding to different promoters

• eukaryotes have several

  • RNA pol I transcribes rRNA

  • RNA pol II transcribes pre-mRNA, small RNAs

  • RNA pol III transcribes tRNA, small RNAs

RNA transcription initiation in prokaryotes

• promoter recognition

• Pribnow box (TATAAT box) in promoter at -10 bp

• Second consensus box (TTGACA) at -35 bp

• sigma factor binds to RNA pol, aids in positioning at TATA box

• RNA/DNA interaction induces strand separation at TATA box

• transcription begins at +1 site

RNA transcription elongation in prokaryotes

polymerase moves downstream building polymer

RNA transcription termination in prokaryotes

• Rho dependent:

  • termination sequence is transcribed, including rut site where Rho protein binds

  • Rho moves 5’-3’ along RNA until it reaches the RNA pol, and separates it from DNA and RNA

• Rho independent:

  • termination sequence is transcribed, reverse complimentary sequences on the RNA interact and form hairpin structure

  • loss of RNA/DNA interaction, freeing RNA

Polycistronic RNA

multiple genes, one promoter

transcribes one long RNA molecule which can encode several proteins

transcription initiation in eukaryotes

• basal TFs bind boxes in promoter

• TF complexes bring in RNA pol

• regulatory TFs bind upstream of promoter, either repressors or activators (RNA pol positively regulated)

RNA transcription termination in eukaryotes

• RNA pol transcribes past the protein coding region into a non-coding region

• hits a PolyA signal and keeps going

• Rat1, RNA binding protein, recognizes polyA signal and cleaves RNA, degrading the rest 5’-3’

• releases pre-mRNA

Polyadenylation

• only in eukaryotes

• Adds a 3’ polyA tail to mRNA

• aids translation

• protects mRNA from degradation (long tail = lasts longer = more protein)

• aids transport to cytoplasm, where translation occurs

capping

• only in eukaryotes

• add a 5’ cap to mRNA

• aids in translation (where ribosome binds)

• protects protein from degradation by 5’-3’ nucleases

• aids transport to cytoplasm

splicing

• only in eukaryotes

• removes introns from protein coding sequences

• required to transfer mRNA out of nucleus

• alternative splicing: multiple proteins from one RNA

tRNA processing

• base modifications

  • tRNA modifying enzymes alter transcribed bases

  • aids secondary structure formation

rRNA processing

• transcript modification

• multiple rRNAs transcribed then processed

• some bases are methylated

• transcript cleaved

start codon for protein translation

first 5’ AUG codon (methionine)

ribosome structure

small subunit: mRNA binding protein

large subunit: enzymatic

translation initiation for prokaryotes

• Shine-Dalgarno sequence recruits ribosome

• binds to complimentary region on rRNA in small subunit

• aligns ribosome with start codon

• initiator tRNA fMet (modified tRNA Met)

• initiation factor 2 (IF2) complexes with fMet and GTP

• IF1 and IF3 complex with small subunit and prevent ribosome assembly until everything is right

• Then, recruits large subunit

• hydrolysis of GTP, release IFs

• ribosome assembles with fMet in place

initiation of translation eukaryotes

• use 5’ cap to recruit ribosome

• small subunit attaches, scans for start codon

• AUG within the Kozak sequence

• fMet initiator tRNA aligns ribosome with correct start codon

• fMet complexes with some IFs, dissociate when fMet binds to start codon

• ribosome assembles with fMet in P site

siRNA

small interfering RNA, initiates degradation of viral RNA