Microbiology test #3

- DNA Replicatin and Gene Expression

DNA Molecule in prokarayotic cell

• two polynucleotides spiraling around an imaginary axis

• asymetric ends

• 5 Carbon end and 3 Carbon end

DNA double helix directionality?

• two molecules (strands) run in opposite directions

• antiparallel

• genetics always goes 5 to 3

The nitrogenous bases in DNA pair up: complementary base pairing

Form hydrogen bonds:

• Adenine bonds Thymine (or Uracil in RNA), has 2 Hydrogen bonds

• Guanine bonds to Cytosine, has 3 hydrogen bonds

In the replication of prokarayotic cells how is the chromosomes different than eukaryotic cells?

• there is only one chromosome

• and the chromosome is circular shape

Bidirectional replication of a circular bacterial DNA molecule

• Starts to unwind at the Origin of replication

• DnaA binds to OriC

• DNA unwinding element, A-T rich because two bonds so it is easier to pull apart, starts to unwind at the Origin of replication

DNA Synthesis

DNA is copied by DNA polymerase (DNA pol)

• in the 5’ → 3’ direction; Initiated by an RNA primer

• leadering strand is synthesized continuously

• lagging strand is synthesized discontinuously; Okazaki fragments

• RNA primers are removed and Okazaki fragments joined

Problems in the copying process: dimerized chorosome

• merge together

• cross-over occurs at ter site

• DNA Gyrase solves this and recombination between and allows to pull apart

• resolution of this by the recombinase

Problems in the copying process: Catenated chromosomes

• interlocked

• DNA Gyrase is used too

• double- stranded break introduced by topoisomerase, allowing passage of the intact chromosome through the gap

• resolution of catenated daughter chromsomes by a topoisomerase

Gene Expression Step 1- Transcription

• polycistronic mRNA often found in bacteria and archaea

• catalyzed by a single RNA polymerase

Polycistronic mRNA

• many genes expressed at the same time

• often found in bacteria and arches

Monocistronic mRNA

• one protein coding region at a time

DNA Gyrase

cuts backbone of DNA

DNA Helicase

unwinds and separates double stranded DNA or RNA into single strand

DNA Primase

lays down the primer

Leading strand

synthesized continuously in the way of replication

Lagging strand

synthesized discontinuously; Okazaki fragments

Start codon for gene expression

AUG

How is the process finished?

RNA primers are removed and Okazaki fragmenets joined

Stop condon for gene expression

UAA, UAG, UGA

Archeal genome expression

• promoter architecture and RNA polymerase structure more similar to eukaryotes than bacteria

Transcription molecular participate: DNA template

the template strand is read 3'→5', and the RNA is made 5'→3'. The coding (sense) strand has the same sequence as the RNA, with T replaced by U.

Transcription molecular participate: RNA polymerase core enzyme

a single enzyme in bacteria that synthesizes the RNA.

Transcription molecular participate:Sigma factor

joins the core enzyme to form the holoenzyme. It helps recognize the promoter and is released after initiation.

Transcription molecular participate: Ribonucleoside triphosphates

the building blocks added to the 3' end of the growing RNA.

Transcription molecular participate: Termination factors

NusA (intrinsic termination) and rho protein (rho-dependent termination).

Important sequences: Promoter

the site where RNA polymerase binds. In bacteria it has a -35 sequence, a 16-18 bp spacer, and a -10 sequence (Pribnow box), followed by the +1 transcription start site. Archaeal promoters are more eukaryote-like, with BRE and TATA boxes and an Inr element.

Important sequence: Terminators: Intrinsic (factor-dependent)

the RNA polymerase pauses in an A-rich region

• RNA stops transcription

Important sequence: Terminators: stem loop

forms in the RNA, and the weak U-A bonds of the RNA-DNA hybrid let go.

Important sequence: Terminators: Rho dependent

rho binds the rut site in the RNA, chases the polymerase, and uses its RNA:DNA helicase activity to separate the hybrid at the terminator.

• uses protein

The genetic code

• mRNA is translated into amino acid sequence using a code

• codons and anticodons

• the code is redundant, non-ambiguous, and universal

tRNA sturucture: Anticodon

first and second position allow for specific pairing with codon; at thrid position, wobble base pairing allows for loose pairing→ less tRNAs than codons are needed

tRNA structure: Amino acid acceptor end

each tRNA specific for a single amino acid and corresponding tRNAs

Ribosomes

site of translation

Coupled transcription/translation

• happens in bacteria bc no nucleus

• transcription and translation can occur simultaneously

16S rRNA

• Shine Dalgarno binding

• bond in small subunit

• helps the bacterial ribosome recognize and postion itself at the translation start site

23S rRNA

• Ribozyme for peptide bond formation

• large subunit

• catalyze the formation of peptide bonds between amino acids

Initiation of Translation

• fMet-tRNA used and binds start codon

• 16S rRNA binds mRNA leader (S-D)

• mediated by IFs

• 1 GTP energy used

Elongagtion of Translation

• New tRNA binding

• transpeptidation

• translocation

• mediated by EFs

• 2 GTP energy used

Termination of Translation

• stop codon reached

• mediated by RFs

• RFs recognize stop

• diassembly

• uses 1 GTP again

Operon molecular components:

operon

is a group of bacterial genes regulated together so the cell can control the production of proteins needed for a particular function.

How are operons used for regulation — inducible vs. repressible?

Inducible operon- usually is silenced (no gene expression), quite all the time, catabolic needs

Repressible operon- active, is the one usually turned on, anabolic needs

both: negative regulation, based on activity of repressor protein

Describe in detail how catabolite operons like the lac and ara operons function.

• three structural genes coding for lactose uptake and metabolism

• lac repressor (lacl) binds operator and inhibits transcription

• enzymes normally not produced unless lactose present

I- mutant

the repressor protein is altered or absent and cannot bind to the operator region