Microbio exam 2 bacterial genetics

DNA in bacteria

2 types : chromosomal and plasmid

DNA→ RNA → protein

blueprint for protein synthesis.

double helix

anti parallel strand 5’-3’(new) and 3’-5’(template)

nucleoside

component of DNA made of ribose/deoxyribose sugar with purine or pyrimidine (nucleobase+ pentose sugar)

nucleotide

nucleoside with one or more phosphate

OH groups tells weather its DNA or RNA

DNA structure

hydrogen bonds between anti parallel strands.

phosphodiester bonds in strands (stronger thatn hydrogen bonds

DNA replication

circular chromosomes, bidirectional 5’ →3’

semi conservative- each stand in DNA molecule serves as a template for a new strand

initiation, elongation, termination

initiation, DNA rep

replication begins at origin (oriC) of chromosome

A-T rich (easier to separate than GC)

DnaA binds oriC at DnaA box

requires energy

consensus sequences

replicaiton bubble proteins

DnaA recongnized by DnaB (helicase) and unwinds DNA.

2 helicases onto two DNA strands travel in opp directions, two rep. forks created

binds DnaA-oriC using energy from ATP hydrolysis

single stranded binding proteins

strands must be spereated during replication

Toposomerase II (gyrase)

relieves supercoiling and relaxes DNA

control of initiation

methylation of oriC

cells can tell difference between fully methylated and hemi (one strand is methlyated)

SeqA binds to hemi methylated, when removed new strand is methylated and new round of replication can be initiated. new strand will become fully methylated if binded by DNA denosine methylase (Dam)

DnaA binds to fully methylayed

elongation, DNA rep

catalyzed by DNA pol III

DNAp adds nucleotides (polymerase I and III important during replication)

low error rate bcs DNA polymerase is highly accurate in its ability to proofread and repair

priming during elongation

NEEDS RNA poly (DNA primase) to create primer for DNA p III. primase creates free 3’ OH for elongation

leading strand

DNA p III extends 5’→3’, continuous

lagging strand

okazaki fragments (shorts sections of DNA created during DNA rep)

read opposite

DNA pol 1 removes RNA primer and replaces it with DNA

DNA ligase fills gaps for phosphideister bonds

RNA primers

binding site for DNA polymerase to initiate DNA replicaiton.

initiate synthesis for Okazaki fragment, replaced by DNA. DNA ligase then fills gaps between fragements

low error rate for dna rep

A-T paris weaker and easier to separate than G-C

DNA pol I and III exonuclease (remove incorrect nucleotides)

mismatch repair system

termination, DNA rep

bidirectional- forks meet at 180 from oriC, ter sites

tus protein bind at ter site and block DnaB

toposiomerase IV breaks strands and resolves linked chromosomes

growth rate

determines number of chromosomes per cell

gene dosage (# of certain gene in a genome)

genes closer to oriC encode produced required in higher amounts

DNA vs RNA

RNA contains U instead of T

RNA contains ribose sugar instead of deoxyribose

mRNA single stranded

coding strand DNA

5’ →3’

mRNA

5’ →3’, encodes proteins

templete strand DNA

3’ →5’

rRNA (ribosomal)

forms core ribosome

tRNA (tranfer)

adapters that bring AAs to mRNA during translation

small regulatory RNA/micro RNA

non coding, regulate gene expression

transcription

DNA copied and RNA (mRNA) made.

done by RNA polymerase, DNA unwinds and rewrites to mRNA (which can translate for proteins)

ATP needed

intitiation, elongation, termination

initiation, transcription

need RNA poly, sigma, promoter

holoenzymes binds at promoter where sigma factor is, DNA unwound (open complex)

RNA polymerase

during initiation of transcription synthesizes RNA

core enzyme 5 subunits

core + sigma = holoenzyme (6 subunit)

sigma factor

help identify location, increase RNA polymerase for specific DNA seq promoter

different for different promoters

promoter

specific sequence on DNA

RNA polymerase holoenzyme

binds to promoter region of DNA through sigma factor

recognizes -35 and -10 position to the start transcription site

elongation, transcription

sigma falls off and recycled

RNA polymerase synthesizes 5’ →3’

slower than DNA rep

termination, transcription

either Rho independent (instinsic) or Rho dependent

Rho independent (instinsic)

most common termination of transcription

G-C rich stem loop structure bonded by hydrogen bonds

destabilizes open complex, RNA polymerase knocked off

followed by Us

Rho dependent

protein Rho factor.

binds to MRNA at rut site and unwinds RNA DNA complex, RNA polymerase dissociates

translation

mRNA into protein

5”→3’

energy intensive,

need ribosomes and charged tRNA (bring amino acids)

intitation, elongation, termination

prokaryotic ribosomes

ribosomes bind mRNA to start. promoter region

70s riboosomes (30S and 50S)

tRNA in translation

contain amino acids and deliver to ribosomes

-charged: bound to amino acid (amino acyl tRNA)

-uncharged: not bound

charged tRNA

synthetase enzyme adds amino acid

can proofread

brings amino acid from cytoplasm to ribosome

which amino acid added during translation?

based on genetic code

codon (sequence of 3 bases)

codons with diff seq can code for same amino acid

ex: codon CGA in triplet code

wobble

loose base pairing, tRNA anticodon can pair with codons that differ

initiation, translation

defined location on mRNA

AUG codon (start codon) fmet bacteria, 30S + 50S subunits

IF1, IF2, IF3 initiation factors

GTP (energy)

shine dalgarno sequence

ribosome binding site

before the AUG start codon

elongation, translation

ribosome has 3 binding sites for tRNA

A (aminiacyl, acceptor) tRNA binds

P (peptidyl, donor) transpeptidation

E (exit)

N to C teminus

transpeptidation reaction

peptidyl transferase

peptide bonds formed between amino acids

between A and P sites

translocation

empty tRNA move from P to E site to exit

tRNA with growing chain moves A to P

new tRNA binds to A

termination, translation

UAA, UAG, UGA are stop codons

release factors 1,2 ,3

final peptide bond, peptide released

ribosomes dissociate

protein processing

1.folding : spontaneous or with chaperone

2.modifications

• remove fmet

• add sugar and lipids

3.transport of protein: if required

(inside to outside is secretion

SEC pathways

cytoplasmin proteins hydrophillic. signal seq on N terminus

signal peptidase and peptide, bring protein across cytoplasmic membrane