MBI 201 Exam 3

Genome

Entire set of genetic information in an organism, including all organism’s genes, whether they’re found on a single chromosome or distributed among multiple genetic elements.

A genome does not include…

RNA viruses, microbial genomes are encoded by DNA

Why is DNA ideal information storage molecule?

Because it is stable, mutable and replicable

In 1928 Griffith discovered…

That he could kill mice with a live and harmless strain of bacteria but only if he coinjected the mice with dead cells from a separate pathogenic strain (didn’t know it was connected with DNA).

In 1944, Avery demonstrated…

that DNA, and not protein or RNA, was responsible for transforming Griffith’s harmless strain of bacterial cells into killers

In 1952, Hershey and Chase showed…

that viral DNA, and not viral protein, was injected into host cells to produce new viruses.

Generally, the simpler the organism,

the smaller it’s genome

Most sequenced bacterial genomes consist of

a single circular chromosome (90%)

Noncoding content/regulatory sequences

promoters and enhancers

Coding vs noncoding genes in prokaryotes vs eukaryotes

In prokaryotes it is very dense with very little noncoding areas, eukaryotes is the opposite with a surplus on noncoding areas.

genes

the coding content in a chromosome in broken up into functional units

purines

Adenine and guanine

how many rings do purines have?

two

pyrimidines

cytosine and thyamine

how many rings do pyrimidines have?

one

what is a nucleotide made up of?

Nitrogenous base (A, G, C, & T), deoxyribose sugar, phosphate group

How are nucleotides in a nucleic acid held together

covalent bond called phophodiester bond

How are nucleic acids held together?

hydrogen bonds, which are overall very weak but strong in numbers

DNA complementary meaning

A pairs only with T (with two hydrogen bonds)

G pairs only with C (with three hydrogen bonds)

The sequences on one strand dictates the sequences on the other

DNA antiparallel meaning

the phosphodiester backbones of two complementary molecules orient themselves in this order (5’-3’ & opposite).

sequence

order of DNA, dictates the function of DNA, no biochemical constraint to this

Grooves

DNA is twisted into a double helix that has two ____ (one major one minor). These give DNA-binding proteins access to base sequences buried in the center of the molecules. DNA-binding proteins can interact w/ specific base sequences without the strands being separated.

Bacterial chromosomes

super compacted, 4.6 million base pairs, stretched out, it is 1,500 times longer than the cell itself

How much dry mass does the chromosome make up of it’s self?

3-4%

Structure of supercoils

Proteins are twisted and then looped and anchored to histone-like proteins

Supercoiles

loops over/under twisted and _____ in order to pack DNA into a series of loops or domains

Positive supercoils

DNA is overwound - twisted in the direction of the inherent twist of the helix

Negative supercoils

DNA is underwound- Twist is against the direction of the inherent twist of of the helix

What kind of supercoils do most cells DNA contain?

Negative

Why do most cells have negatively twisted supercoils?

The two strands of DNA are easier to separate

Why do archea have positively twisted supercoils?

Living in acid at high temperature, this allows them to protect their DNA

Topoisomerases

Catalyze the formation and relaxation of supercoils

Process of producing a supercoil

1. produce a double-strand break in the circular chromosome

2. pass an intact region of the DNA through the break

3. Seal the ends to generate a twist in the chromosome

Semiconservative DNA

each new double helix is composed of one parental strand and one newly synthesized strand

How many origin sites are found in bacterial DNA

only one - oriC

What happens after initiation of replication?

a bubble forms pulling the two strands apart

What happens after replication bubble is formed?

contains two replication forks that move in opposite directions around the chromosome to replicate it in a bidirectional fashion.

How does replication end?

At defined termination (ter) sites located opposite origin

During replication, do many species start a new round before even finishing the first?

yes

oriC

The start of DNA replication is controlled by the binding of a specific initiator protein to the origin.

DnaA

Triggers initiation by displacing SeqA

SeqA

inhibits replication at inappropriate times

Replisome

replication machine

DnaA function

melts the DNA and begins to assemble a replication machine

DnaC

helicase loader - loads DnaB onto each single-stranded template

DnaB

helicase - unwinds the double helix progressively to expose the individual strands and recruits DnaG

DnaG

primase - synthesizes a short RNA primer complementary to a region on each template strand

the replisome is completed by this polymerase

DNA pol III

Clamp proteins

added to keep pol III engaged with the DNA

what direction is new DNA always synthesized in?

5’ to 3’

what direction does DNA polymerase alyways read/move along DNA in?

3’ to 5’

leading strand

Allows continuous synthesis toward the replication fork

lagging strand

Requires discontinuous synthesis away from the replication fork

Okazaki fragments

these fragments are progressively stitched together to make a continuous unbroken strand from lagging strand

DNA gyrase

As synthesis proceeds, operates ahead of each fork to relieve the torsional strain caused by helicase activity.

RNA primers removal

by RNase H or the exonuclease activity of DNA polymerase I

RNA primers function

short, single-stranded segments of ribonucleotides that provide a necessary starting point (3'-hydroxyl group) for DNA polymerase during DNA replication

DNA pol I

after removing rna primer, synthesizes a DNA patch using the 3' OH end of the preexisting DNA fragment as a priming site

DNA ligase

Repairs the remaining phosphodiester nick using energy from NADH (bacteria) or ATP (eukaryotes).

TUS

(terminus utilization substance)

what does TUS do?

binds to these sequences and ensures that the polymerase complexes stop and do not continue replicating DNA.

Catenane

two daughter molecules (sister chromosomes) appearing as a pair of linked rings

Topoisomerase IV

Separate’s sister chromosomes

Why must catenane be resolved?

the two rings must be unlinked—so that the sister chromosomes can segregate prior to cell division

transcription

reading a DNA template to make an RNA copy

translation

decoding the RNA to assemble protein

Difference between RNA and DNA stability?

Presence of a hydroxyl group instead of a proton at the 2’ position of the ribose makes it less stable on rna

RNA polymerase

enzyme that carries out transcription by making RNA copies of a DNA template strand

How does RNA pol replicate?

It does this not by copying the entire genome, but discrete segments of it at specific locations, corresponding to genes

What is RNA pol made up of?

Core polymerase (alpha2, b, b’), sigma factor

holoenzyme

core polymerase and sigma factor together

sigma factor function

helps the core enzyme detect a promoter

promoter

DNA sequence that signals the beginning of a gene or group of genes (operon), typically sigma-70

consensus sequence

helps identify important functional areas, such as binding sites for proteins, gene promoter regions . At the -10 and -35 positions.

First phase of transcription

Initiation: RNA pol holoenzyme binds to the promoter followed by separation of the DNA strands and synthesis of the first nucleotide of the RNA

second phase of transcription

elongation: the RNA chain is extended

third phase of transcription

termination: RNA pol detaches from the DNA, after the transcript is made

Operon

group of genes (one to many; two in the diagram below) that get transcribed on the same mRNA molecule (under the control of the same promoter)

Relationship between mRNA and the two strands of DNA during transcription?

the mRNA is complementary to the template strand and identical in sequence to the sense strand (except T in DNA is U in RNA).

APE

70S ribosome harbors three adjacent binding sites

A(acceptor site)

Binds incoming aminoacyl-tRNA

P (peptidyl-tRNA) site

harbors the tRNA with the growing polypeptide chain

E (exit) site

binds a tRNA recently stripped of its polypeptide

initiator tRNA

used for first codon, in bacteria it carries N-formylmethionine

Intiation of transcription

1. RNA pol holoenzyme forms a closed complex w/ DNA where it “recognizes” a promoter sequence (w/ help of sigma factor)

2. closed complex must become an open complex through unwinding of 1 helical turn

3. RNA pol in open complex becomes tightly bound to DNA and so begins transcription (no primer needed)

Elongation of transcription

1. RNA pol moves along template (slower than DNA pol 3)

2. newly synthesized material (RNA) is read 3’ - 5’ and produced 5’ - 3’

3. unwinding DNA ahead of complex → transcription bubbles

4. super coils removed ahead by DNA topoisomerase

5. this process proceeds and the hybrid DNA/RNA strand is unstable so DNA put back together and RNA is “diplaced”

Termination of transcription - Rho dependent kiss of death

1. near 3’ end a C-rich sequence is transcribed followed by sequence that causes a pause in elongation

2. protein Rho climbs onto RNA at C-rich sequence and then it uses ATP hydrolysis to crawl along RNA toward the RNA pol

3. when Rho reaches and touches RNA polymerase, it releases the RNA

Termination of transcription - Rho independent blocked!

1. requires GC rich region of RNA and 4-8 consecutive U residues at end of region to be transcribed

2. immediately after synthesis, base pairing in GC segment causes the RNA to fold into a hairpin loop which binds to NusA

3. NusA prevents RNA pol from form moving past

4. nearby seq of all U’s is base paired weakly w/ DNA and doesn’t take long for RNA & DNA to disassociate → RNA pol is released

messenger RNA (mRNA)

encodes proteins

ribosomal RNA (rRNA)

forms ribosomes

transfer RNA (tRNA)

• shuttles amino acids

• single stranded decoder molecule

• contains 2 functional region (anticodon and 3’ end)

• must be charger before encountering ribosome

small RNA (sRNA)

regulates transcription or translation

tmRNA

free ribosomes stuck on damaged mRNA

catalytic RNA

carries out enzymatic reactions

anticodon

H bonds with mRNA codon specifying an amino acid

3’ (acceptor) end

• binds specific amino acid

• contains a lot of modified bases

aminoacyl tRNA synthetases

• charge tRNA for translation

• recognize it’s own tRNA

• each tRNA has own set of sites that match specific synthetase

• ATP hydrolysis to AMP and pyrophosphate powers this energetically unfavorable charging

  • energy from atp hydrolysis is stored in bond between tRNA & amino acid to be used to form peptide bond

Initiation of translation

1. ribosomes recognize start codon on mRNA to begin

2. special tRNA initiator is used for this first codon

   • N-formylmethionine AA

3. Large and small subunits separate from each other

4. mRNA interacts favorably with 30s subunit

   • start codon is positioned in P site and initiator tRNA comes along

5. Initiation factors(IF1, 2, 3) make sure that it’s really initiator tRNA

   • then 50s subunit binds

   • IF2 confirms that everything is interacting correctly, hydrolyzes GTP and proceed

*if IF2 doesn’t allow to proceed → everything falls apart and starts over

Elongation of translation

*next codon is in the A site

1. the corresponding tRNA, carrying the next amino acid, fits in there

2. elongation factor makes sure it’s the right tRNA

   • Verifies twice, hydrolyzes GTP and process continues

3. amino acid in P site is transferred to the AA in A site, making peptide bond

   • uses energy that was stored in bond when tRNA was charged

4. another elongation factor hydrolyzes GTP to help the ribosome move to next codon in 3’ direction

5. now older uncharged tRNA is in the E site and growing peptide is on the tRNA int the P site

   • when tRNA goes in A, one leaves through E site