Ch. 13 + 16: Bacterial Genome and Genome Variations

What is DNA the template for

DNA replication and transcription

replication

DNA → DNA

create 2 identical copies of DNA molecule

• DNA polymerase

• DNA template read 3’ → 5’

• DNA synthesized 5’ → 3’

transcription

DNA → RNA

copying DNA sequence into mRNA molecule

• RNA polymerase

• DNA template read 3’ → 5’

• mRNA synthesized 5’ → 3’

translation

RNA → Protein

use mRNA sequence to build protein

• ribosome

• mRNA codons read 5’ → 3’

• polypeptide synthesized N → C

  • info on maturation at N terminus

  • at least one sulfur containing AA in sequence

replication, transcription, and translation in EUKARYOTES

replication and transcription: nucleus

translation: cytoplasm

replication, transcription, and translation in PROKARYOTES

replication, transcription, and translation occur in cytoplasm

What carries out DNA replication

DNA polymerase - few errors; 1 in a million

What carries out transcription

RNA polymerase (enzyme that synthesizes RNA from DNA)

What is mRNA the template for

proteins

What carries out translation

ribosomes

What does reverse transcriptase do

RNA into DNA (specifically cDNA)

• identified from viruses

can you turn protein into RNA

no

degeneracy

Griffith

transformation test

• demonstrated that non-virulent bacteria can be transformed into virulent forms

What did the transformation test show

the change of nonvirulent organisms into virulent ones via transformation

• combining dead versions of bacteria with living versions of nonpathogenic bacteria → pathogenic bacteria

What traits did the transformation show

S strain and R strain

S strain

smooth, pathogenic, has capsule

R strain

rough, not pathogenic, no capsule

What did Avery-Macleod-McCarty do

identify DNA as the transforming factor (when DNA was destroyed, transformation did not occur, not pathogenic)

• treating type S DNA extract with DNase didnt lead to transformation of Type R cells

What did Hershey-Chase experiment prove

that DNA is the genetic material because only the phage DNA entered the cell

• utilized radioactive elements (35S and 32P) and bacteriophage to show that DNA is genetic material

What did the Hershey-Chase experiment use as a model

bacteriophage T2 (DNA virus)

Details of the Hershey-Chase experiement

DNA was labeled with 32P
Protein coat was labeled with 35S
Only the labeled DNA entered the cell

Why did only DNA enter the bacterial cell

because phages leave the coat outside

What would’ve happened if the Hershey-Chase experiment was conducted with a mammalian virus

both the protein coat and DNA would’ve entered the cell

What are nucleic acids

polymers of nucleotides

What are nucleic acids linked by

phosphodiester (covalent) bonds

How do DNA and RNA differ

RNA: Uracil, OH on C2, single-stranded
DNA: Thymine, H on C2, double-stranded

strand structure of viruses (nonliving)

can be either double stranded RNA or single-stranded DNA

nucleoside vs nucleotide

nucleoside: sugar and nitrogenous base
nucleotide: sugar, nitrogenous base, phosphate

• may be mono, di, or triphosphate

What are the purines

adenine and guanine (PURe As Gold)
double ring

Pyrimidines

cytosine, uracil, and thymine
single ring

identify nucleoside vs nucleotides / DNA vs RNA / purine vs pyrimidine

• 1: DNA, nucleoside, purine

  • note the H on carbon 2 (DNA), and lack of phosphate

  • double ring: purine

• 2: DNA, nucleotide, pyrimidine

  • note the H on carbon 2 (DNA), and presence of phosphate

  • single ring: pyrimidine

• 3: RNA, nucleotide, pyrimidine

  • note the OH on carbon 2 (RNA), and presence of phosphate

  • single ring: pyrimidine

• 4: RNA, nucleotide, purine

  • note the OH on carbon 2 (RNA), and presence of phosphate

  • double ring: purine

What holds together the double-stranded helix

H-bonds

DNA strands are…

anti-parallel and complementary

what direction is DNA read

3’ to 5’

which is the leading and lagging strand of DNA

leading: 3’ (continuous)

lagging: 5’ (discontinuous)

Okazaki fragments

discontinuous pieces of newly synthesized DNA that form on the lagging strand during DNA replication

What direction do nucleotides get added to DNA

5’ to 3’

how to describe DNA replication

semi-conservative - one old and one new strand

how many times does DNA replicate a genome during a replication cycle

once

where does phosphate bind on DNA

carbon 5

What holds together the back bone of DNA

phosphodiester (covalent) bonds

How many bonds do A-T have

2 H-bonds

what kind of area does RNA polymerase bind in

areas that are A/T rich

• weaker bonding makes the area easier to unwind and separate for transcription initiation

• broken apart by heat and enzymes

• breaking apart with covalent bonds requires a lot of energy

How many bonds do C-G have

3 H-bonds

Major Groove

contains the most information and you can read the nucleotides

What is a ribozyme

an RNA enzyme

carries genetic information

Most RNA molecules are…

single stranded

• tertiary structure gives it function

contains both purines and pyrimidines

used as template for DNA

What virus contains double-stranded RNA

rotavirus

What are polypeptides built from

amino acids (monomer)

What bond holds together polypeptides

peptide bonds (covalent)

How are polypeptides synthesized

Amino-terminus (N) to carboxyl-terminus (C)

what gives polypeptides function

3D structure

• called a protein once it is properly folded and functional

proteins are made up of how many polypeptides

single or multiple

how are proteins denatured

by pH and temperature; breaks nonvocalent bonds

• loses secondary, tertiary, quaternary structure

4 levels of structure of proteins

Primary, Secondary, Tertiary, and Quaternary

Primary proteins

covalent bonds between amino acids

per HW: a sequence of a chain of AA

Secondary proteins

alpha helix and beta sheets, H-bonds

structures formed via hydrogen bonding between carbonyl and amino group of the peptide backbond (alpha helices and beta sheets)

Tertiary proteins

noncovalent bonds + disulfide bridge (covalent)

3D folding driven by side chain interactions

Quaternary proteins

Noncovalent bonds + disulfide bridge
ex. Insulin

structure consisting of multiple subunits coming together

alpha carbon is attached to

a hydrogen, R group, amino group, and carboxylic acid group

categories of amino acids

polar, nonpolar, or charged

important AA to note

methionine (AUG) and cysteine (UGU, UGC)

What does methionine have

sulfur to identify proteins

it is the 1st amino acid added to polypeptide chain (AUG)

• every polypeptide should have a methionine

What does cysteine have

sulfhydryl group

What allows disulfide bond/bridges to be made

sulfhydryl group on cysteine

disulfide bridge is between 2 cysteines in the same polypeptide

tertiary structure

disulfide bridge is between 2 cysteines in different polypeptides

quaternary structure

what shape is DNA in most bacteria

circular

what direction is bacterial DNA replication

bidirectional from oriC to ter site

bacterial DNA replication consists of

two replication forks: bidirectional replication from oriC

• Works in both directions from OriC → forks meet at Ter

single replicon: one chromosome; one beginning and end for replication

DNA pol III, single oriC site, topoisomerase

what is a replisome

complex made of polymerase, nucleotides, and topoisomerases moving in both directions; carries out DNA replication

• DNA polymerase III: adds NT in 5’ → 3’ direction

• dNTP (deoxynucleoside triphosphates): raw materials (A, T, G, C building blocks) added to growing DNA strand

• xerCD: separates duplicated chromosomes at the end of replication (ter)

topoisomerases

Relieve supercoiling ahead of the replication fork by cutting and rejoining DNA

bacterial RNA polymerase structure

Core enzyme

• 5 subunits

Sigma factor (σ)

core enzyme + sigma factor = holoenzyme (binds to DNA at promoter)

core enzyme

• responsible for elongation phase

• Made of 5 subunits: two α, β, β′, ω

• β and β′: bind to DNA and form the catalytic center

  • allows RNA polymerase to bind to DNA

• makes RNA but can’t start alone

sigma factor

like a targeting system

• recognizes NT sequence

  • finds the promoter on DNA and start transcription

  • After initiation, sigma falls off — not needed for elongation or termination

• Most common one: σ⁷⁰ (used in normal growth conditions)

holoenzyme

core enzyme + sigma factor = holoenzyme (binds to DNA at promoter)

functional transcription started

what is a promoter

upstream DNA region;

recognized by RNA polymerase with help of sigma factor → strands separate and RNA synthesis begins

• not transcribed

RNA polymerase reads DNA 3′ → 5′, synthesizing mRNA 5′ → 3′

promoter features

-10 region: A/T rich (easy to separate d/t 2 H bonds)

-35 region: upstream recognition site for RNA polymerase

+1 site: where transcription begins

three types of RNA

mRNA, tRNA, rRNA

mRNA (messenger RNA)

carries message (coding instructions) for protein synthesis

• translated into AA

tRNA (transfer RNA)

carries AA to ribosome during translation (protein synth)

has anticodon that pairs with mRNA

rRNA (ribosomal RNA)

components of ribosomes (rRNA + protein)

• has secondary hairpin loop structures

are tRNA and rRNA translated

no, they are functional RNAs

their function depends on the NT sequence and 3D shape

in bacteria, transcription has…

• a defined start site (promoter)

• ends at termination site

genes

basic unit of genetic information

non-protein coding genes

genes that code for tRNA and rRNA; do NOT get translated

what is transcribed together in bacteria

tRNA and rRNA

how are tRNA and rRNA removed

by ribozymes or ribonucleases

structure of rRNA/tRNA gene transcription unit

• Promoter – RNA polymerase binding site

• Leader – non-coding region before coding region

• Coding region – the actual tRNA or rRNA gene

• Trailer and Terminator – sequences downstream of coding region

These segments often contain spacer regions - removed during processing

post-transcriptional modification is done by

ribozymes: RNA molecules with catalytic activity (self-cleaving)

• made of nucleotides

• cut RNA at specific sequences, no protein needed

ribonucleases (RNases): RNA or nucleotides with proteins

• cut RNA or RNA-protein complexes

• remove spacers if multiple tRNAs are in pre-processed transcript

direction of RNA polymerase reading and synthesis

• Reads DNA 3′ → 5′

• Synthesizes RNA 5′ → 3′
  ➤ Always adds new nucleotides to the 3′ end of the growing RNA

protein-coding genes

structural genes that encode polypeptides

• via mRNA → translation into polypeptides

anti-sense (template)

DNA template strand that directs mRNA synthesis

• complementary to sense and mRNA

sense (coding)

complementary DNA strand that has the same NT sequence as mRNA (except T instead of U)

• RNA is a copy of the sense strand but made off the antisense

leader sequence

5’ untranslated region (UTR) of mRNA before coding region

contains shine-dalgarno sequence

Shine-Dalgarno sequence

helps ribosome bind for translation to begin; not translated

polycistronic mRNA

one mRNA that encodes multiple proteins

prokaryotes

monocistronic mRNA

one mRNA encodes one protein

eukaryotes

operon

• makes polycistronic mRNA

• encodes lac permease

• contolled by a single promoter but includes multitude of genes downstream

• Contain genes for proteins with similar or same functions

coupled transcription and translation

in prokaryotes (bacteria and archaea)

• this is possible because both happen in the cytoplasm