Bacterial Gene Expression

The Central Dogma is the flow of genetic information

DNA {transcription}→ RNA {translation}→ protein

DNA= instructions (recipe)

RNA= messenger (copy of recipe)

Protein= function (actual dish)

DNA

Stores genetic info

determines genotype

RNA

Intermediate carrier

Transfers info from DNA

Protein

Does the work (enzymes, structure, transport, function)

determines phenotype

Transcription

Making RNA from DNA

The process of when genetic info in DNA is copied into RNA

Monocistronic and Polycistronic

Monocistronic

1 mRNA codes for 1 protein

Polycistronic

1 mRNA codes for multiple proteins

Translation

The process where ribosomes read mRNA and use it to build a protein

Transcription and Translation coupled occur where in a bacterial cell?

Cytoplasm

RNA Polymerase

An enzyme that reads a DNA template and builds a complementary RNA strand during transcription

Synthesizes in the 5’-3’ direction (like DNA)

Can start on its own, NO PRIMER

Sigma Factor (σ)

A protein that helps RNA polymerase find the promoter, bind to it, and start transcription in bacteria

Will fall off after the core puts on about 10 bases

Recognizes -35 and -10 regions

Sigma + Core =

Holoenzyme

Promotor

Transcription start site

Steps to Transcription

Initiation

Elongation

Termination

Prokaryotes vs Eukaryotes

Bacteria: transcription and translation happen at the same time and in the cytoplasm, operons are multiple genes together, no introns or exons they just transcribe what they have

Eukaryotes: have introns and exons, need splicing, introns removed and mRNA is formed

Bacteria

Simple, fast, together

Eukaryotes

Complex and separated

Coding Strand

DNA strand that matches the RNA sequence (except T replaces U)

Template strand

The DNA strand used as a guide to make RNA during transcription

-Inverse of the mRNA

Transcription Initiation

The first stage of transcription where RNA polymerase binds to the promotor (with help from sigma factor in bacteria) and begins RNA synthesis

Transcription Elongation

The stage in transcription where RNA polymerase moves along the DNA template and adds nucleotides to lengthen the RNA strand

Transcription Termination

The stage in transcription where RNA polymerase reaches a termination signal and releases the newly made RNA transcript

Two ways:

-Rho-dependent

-Rho-independent

Rho-dependent

Type of transcription termination in bacteria that requires the Rho protein to unwind the RNA-DNA hybrid and release the RNA transcript

Uses Rho protein

1. A protein called Rho binds to the newly made RNA strand

2. Rho moves along the RNA toward RNA polymerase using energy (ATP)

3. RNA polymerase pauses at a termination region on the DNA

4. Rho catches up to RNA polymerase

5. Rho helps separate the RNA from the DNA template, stopping transcription

Rho-independent

1. RNA polymerase transcribes a DNA sequence that forms a GC-rich hairpin loop in the RNA

2. Right after the hairpin, there is a stretch of Us in the RNA

3. The hairpin causes RNA polymerase to pause

4. The weak A-U bonds (between RNA and DNA) break easily

5. The RNA strand detaches, ending transcription

There are 3 types of RNA

mRNA

rRNA

tRNA

mRNA

Carries genetic information to ribosome that encodes for a protein

rRNA

RNA associated with the ribosome

5s rRNA (associated with the large subunit)

16s rRNA orients/positions the ribosome on the mRNA (associated with the large subunit)

23s rRNA peptidyl transferase activity (associated with the small subunit)

tRNA

Brings amino acids to the ribosome during protein synthesis

-Dihydrourine loop → involved in charging tRNA

-Pseudouridine loop → involved in ribosome recognition

-Anticodon loop → involved in mRNA recognition; recognizes codons in mRNA 3 bases at a time

-3’ end → amino acid attached here to the rRNA

Aminoacyl-tRNA Synthetases

Enzymes that catalyze the attachment of amino acids to tRNA

-There is a specific enzyme for each amino acid

No proofreading in the ribosome, so this is the step that adds specificity

less specific for tRNA (can recognize tRNA for each amino acid)

Genetic Code

Triplet code (codon = 3 bases on mRNA)

Anticodon = matches on tRNA

Start codon

Code is degenerate (specific to certain amino acids)

Base wobble

Start Codon

AUG aka met AKA fMet in bacteria! (N-formyl methionine, first amino acid)

different than other AUG

Bacteria are more flexible than eukaryotes and other start codons sometimes

Base Wobble

3rd base is flexible/wobbly!

A single tRNA can recognize multiple codons → less tRNA needed in the cell

Allows for more wiggle room

Each codon codes for a specific amino acid, but some amino acids can have multiple codons for one amino acid

Steps to Translation

1. Ribsosome binds mRNA

2. tRNA brings amino acids

3. Peptide bonds form

4. Protein grows

ALSO HAS Initiation, Elongation, and Termination

Shine-Dalgarno Sequence

A sequence that is rich in A and G that comes before the actual start codon

Helps it line up with the chromosome (aligns the ribosome with the start codon)

16s rRNA is crucial for this alignment

Ribosome

A cellular structure that reads mRNA and builds proteins by linking amino acids together

E-site (Exit site)

Where tRNA leaves after donating its amino acid

P-site (peptidyl-tRNA binding site)

Where peptide bonds are formed in the ribosome

Where Shine-Dalgarno Sequence binds to SSU

A site (Aminoacyl-tRNA binding site)

Accepts new tRNA

Small subunit

Part of the ribosome that binds to mRNA and helps it correctly position the start codon for translation

Large subunit

The part of the ribosome that forms peptide bonds between amino acids during protein synthesis

G proteins

Other proteins that help other than the ribosome (helper proteins)

-Initiation and Elongation factors

-GTP switches from active to inactive states

Translation Initiation

First stage of translation where the ribosome assembles on mRNA and the first tRNA binds to the start codon

1. Ribosome binds to mRNA and looks for Shine-Dalgarno Sequence so it can “Start” and line up with the ribosome

2. Finds the start codon

3. First tRNA enters, methionine enters with tRNA and binds to AUG

4. tRNA sits in the P site, GTP hydrolyses

5. IF3 gets bumped off by IF-1

6. Large subunit comes in and IF-1 leaves

7. Complex is complete and is ready to build a protein

Translation Elongation

Stage of translation where the ribosome moves along the mRNA, and tRNAs bring amino acids that are added to the growing protein chain

Goal: add amino acids 1 by 1

1. brings appropriate tRNA to the A site (via EFU-TU complex)

2. Peptide bond formation → created by 23s ribosomal RNA

3. Translocation → ribosome moves 3 bases toward 3’ end (A to P, P to E, E to outside)

Chloramphenicol

Inhibits peptide bond formation

Erythromycin

Inhibits translocation

Translation Termination

Final stage in translation where a stop codon is reached and the completed protein is released from the ribosome

1. Ribosome hits a STOP codon

2. Release factor binds

3. Protein is released

4. Ribosome falls apart (subunits separate and mRNA is released) IF3 attaches to the small subunit and triggers the separation of the subunits

“Stop, release, disassemble”

-different release factors recognize different stop codons

-peptide gets cleaved off from tRNA in p-site

No tRNA recognizes stop codons…

They recognize release factors!

IF-3

G-protein in bacteria that prevents small and large subunits from prematurely joining, ensuring correct initiation of translation

EFU-TU complex

Bacterial protein that delivers aminoacyl-tRNA to the ribosome during translation elongation