CH.13- Bacterial Genome Replication & Expression

Fred Griffith

Fred Griffith

- introduced living, encapsulated cells to mice and observed that mice died

- introduced living, non-encapsulated cells and heat-killed encapsulated cell to mice and observed mice lived

- introduced both heat-killed encapsulated cells and living, non-encapsulated cells into mice and observed mice died

- able to isolate living, encapsulated cells from dead mice

- discovered transformation

deoxyribonucleic acid (DNA)

a polynucleotide that constitutes the genetic material of all cellular organisms; alternating sugar and phosphate backbone w/ bases attached to each sugar

DNA base pairs

- adenine pairs w/ thymine using 2 H bonds

- guanine pairs w/ cytosine using 3 H bonds

ribonucleic acid (RNA)

a polynucleotide composed of ribonucleotides joined by phosphodiester bonds

RNA base pairs

- adenine pairs w/ uracil

- guanine pairs w/ cytosine

nucleotide

a pentose sugar attached to a nitrogenous base and phosphate group

pyrimidine

nitrogenous bases w/ one ring structures (i.e. cytosine, thymine, and uracil)

purine

nitrogenous bases w/ double-ringed structures (i.e. adenine and guanine)

*Pure As Gold

origin of replication

a site on a chromosome or plasmid where DNA replication is initiated

replicon

a unit of the genome that contains an origin of replication and in which DNA is replicated

replisome

a large protein complex that copies the DNA double helix to form 2 daughter chromosomes

helicase (aka DNA B)

enzyme that is uses ATP to unwind DNA ahead of replication fork

DNA polymerase I

removes primers during DNA synthesis

DNA polymerase III

enzyme that catalyzes the synthesis of DNA; also has an exonuclease proofreading function

primase

- enzyme used to synthesize a RNA molecule complementary to the template to act as DNA polymerase III’s starting point

- produces a 10-base long RNA

single stranded DNA binding proteins (SSBs)

proteins that coat single stranded DNA to prevent it from re-annealing and protect it from damage

topoisomerases

enzymes that change the structure of DNA molecules by transiently breaking one or both strands; relives supercoiling ahead of replication bubble

leading strand

strand of DNA that is synthesized continuously during DNA replication

lagging strand

strand of DNA synthesized discontinuously during DNA replication

Okazaki Fragments

short stretches of polynucleotides produced by the lagging strand

DNA ligase

enzyme that joins 2 DNA fragments through the formation of a new phosphodiester bond

catenanes

circular, covalently linked chromosomes

cistron

segment of DNA that encodes a single polypeptide

transcription

process of synthesizing a mRNA molecule using DNA as a template

stages of DNA Replication

1. initiation

2. Elongation

3. Termination

initiation

RNA polymerase binds to the DNA and DNA starts to unwind

• The 50S ribosomal subunit is bound to the 30S subunit on the mRNA, positioning the fMet-tRNA in the P site

Elongation

RNA polymerase connects nucleotides that base pair w/ the template strand nucleotides, forming RNA

Termination

RNA polymerase reaches terminator sequence, which stops transcription and mRNA is released

transfer RNA (tRNA)

binds to mRNA sequences to carry specific amino acids to a growing polypeptide chain; 70-95 bases long

messenger RNA (mRNA)

carries genetic information to the ribosome, which is used to make proteins

ribosomal RNA (rRNA)

structural component of ribosomes that sometimes acts as a catalyst

sense strand

strand of DNA that is not used as the transcription template

promoter

region of DNA at the start of a gene that RNA polymerase binds to before beginning transcription

leader

a sequence in a gene that lies between the promoter and start codon

Shine-Dalgarno Sequence

Region in mRNA’s leader sequence that initiates translation

Terminator

sequence that marks the end of a gene; stops transcription

operon

in bacteria, the sequence of bases in DNA that contains a promoter and 1+ structural genes

Leader

where elongation begins, is transcribed but not translated later

DNA A

protein that activates initiation of replication by forming replication bubble

coding strand

DNA strand w/ the same sequence as the mRNA

translation

process of synthesizing a polypeptide from the mRNA; P site to E site to A site

polycistronic

an mRNA that has one or more coding regions

anticodon

3 bases on a tRNA that are complementary to the mRNA codon

Aminoacyl (A site)

where a charged tRNA molecule is bound to the ribosome

Peptidyl (P site)

where amino acid is attached to the growing polypeptide sequence

Exit (E site)

where tRNA molecules go before they are released from the ribosome

translocation

movement of the ribosome relative to the mRNA that repositions the A site of the ribosome over the next codon to be translated

stop codons

UAA, UAG, UGA

AUG

start codon

DNA replication

proceeds from 5’ to 3’ direction

DNA polymerase III f(x)

1. synthesize an RNA primer

2. Discard old beta clamp proteins.

3. Load new beta clamps to the next spot on the DNA to be replicated.

4. Tether the template to the core enzyme.

RNA polymerase

catalyzes transcription

bacterial translation beginning

1. initiation factors assist an fMet-tRNA to bind to a free 30S ribosomal unit

2. 16S rRNA portion of the 30S subunit aligns w/ the Shine-Dalgarno sequence in the leader region

addition of amino acids

four GTP are hydrolyzed and two ATPs are required for activation