Lecture 9: Microbial Transcription and Translation

Microbial Genetics

• Nucleic acid

• Deoxyribonucleic acid (DNA)

• Ribonucleic acid (RNA)

• Central dogma: Genes (DNA) -> transcript (RNA) -> protein (function)

Transcription

• The reading of a DNA template to make an RNA copy

• First step of gene expression

• Transcription is guided by un-transcribed DNA regulatory sequences upstream of the gene

RNA Polymerase

• Core Enzyme: RNA polymerase (\alpha\alpha\beta\beta’subunits ) – RNA synthesis

• Sigma factor (\sigma):

  • Only needed for the initiation of RNA synthesis (not elongation)

  • Recognizes promoters by binding to -10 and -35 regions of genes

  • Guide the core enzyme to initiate transcription

• Holoenzyme: core enzyme plus Sigma factor

Sigma Factors

• Guides RNA polymerase to promoter

• Multiple \sigma factors within the cell turn on transcription of different genes, responding to different conditions.

• Transcriptional start site: Usually a purine – A or G

Consensus Sequences of σ70 Promoters

• -35 region: TTGACAT

• -10 region: TATAAT

  • Located 17 ± 1 base pairs (bp) downstream from the -35 region.

Consensus Sequence of σ70 Dependent Promoters

• \sigma70 can be seen as the “housekeeping” sigma factor

• -35 and -10 are in relation to the transcription start site (+1)

Other Sigma Factors

• RpoD \sigma70:

  • Promoter recognized: Most genes

  • Promoter consensus sequence:

    • -35 region: TTGACAT

    • -10 region: TATAAT

• RpoH \sigma32:

  • Promoter recognized: Heat-shock-induced genes

  • Promoter consensus sequence:

    • -35 region: TCTCNCCCTTGAA

    • -10 region: CCCCATNTA

• RpoF \sigma28:

  • Promoter recognized: Genes for motility and chemotaxis

  • Promoter consensus sequence:

    • -35 region: CTAAA

    • -10 region: CCGATAT

• RpoS \sigma38:

  • Promoter recognized: Stationary phase and stress response genes

  • Promoter consensus sequence:

    • -35 region: TTGACA

    • -10 region: TCTATACTT

• RpoN \sigma54:

  • Promoter recognized: Genes for nitrogen metabolism and other functions

  • Promoter consensus sequence:

    • -24 region: CTGGNA

    • -12 region: TTGCA

Initiation of Transcription

1. Sigma factor binds DNA

2. Sigma factor recruits the core enzyme and scans for the promoter region

3. Core enzyme unwinds DNA at promoter (“Open complex”)

4. Sigma factor is released

Transcription Elongation

• Core polymerase synthesizes RNA strand 5’ -> 3’

• Added base is complementary to the template strand

• mRNA has the same sequence as the non-template strand but uses U instead of T

Transcription Termination

• Two termination mechanisms

  • Rho-dependent termination

  • Rho-independent termination

Rho-dependent Termination

• Rho (\rho$$) factor binds to mRNA

• It’s a helicase (unwinds nucleic acids)

• Rho pulls itself to the paused RNA pol

• Breaks polymerase, mRNA off the DNA

Rho-independent Termination

• Polymerase slows at the “pause site”

• GC-rich sequence, forms stem loop

• Series of U residues downstream of pause site

• DNA-RNA U-A base pairs are very unstable

• mRNA breaks off the DNA, polymerase released

Types of RNA

• mRNA—messenger: Used to carry information from DNA to protein

• rRNA—ribosomal (structural, non-coding)

• tRNA—transfer

• Other RNAs regulate transcription

  • sRNA—small RNAs: regulate stability or translation of specific mRNAs into proteins

Translation

• The decoding of RNA into protein

The Genetic Code

• Triplets of nucleotides – codons

  • 61 codons → 20 amino acids (degenerate)

  • 3 stop codons

Open Reading Frame (ORF)

• Contained within mRNA and located between the translation start codon (AUG) and stop codon

• Each transcript has three possible reading frames
  Stop codon in the same frame as start codon

tRNA

• tRNAs bind individual amino acids

• tRNAs have a specific shape – cloverleaf structure

• tRNAs have a 3-base anticodon

  • Base pair to codons in mRNA

• Aminoacyl-tRNA transferases attach amino acid aa to tRNA (“charge” the tRNA)

Ribosomes

• Very large rRNA/protein complexes

• 2 subunits (30S and 50S), 52 proteins, 3 rRNAs

  • 30S “small” subunit – 16S rRNA + proteins

  • 50S “large” subunit – 5S and 23S rRNA + proteins

• rRNA forms the catalytic center of the ribosome’s peptidyltransferase activity

• Bind 1 mRNA + 3 tRNAs

Translation is Coupled in Prokaryotes

• Transcription and translation are coupled in prokaryotes

• There’s nothing separating the two processes!

• Ribosomes bind mRNA while mRNA is still being synthesized

• Multiple ribosomes bind to each mRNA

• Proteins are made super fast

Translation Initiation

• Ribosome-binding site (Shine-Dalgarno sequence) on mRNA allows binding to 30s subunit

• IF3 brings “bottom” of the ribosome (30S) and mRNA together

• IF1 blocks the A site

• IF2 escorts the Formylmethionine tRNA (fmet) to the start codon (AUG)

• tRNA binds directly to P site

• Then the “top” (50S) of the ribosome docks to the “bottom” (30S) subunit

Translation Elongation

• 3 steps:

  1. aminoacyl-tRNA binds to the A site (acceptor site)

  2. peptide bond formed between the new aa and the growing peptide chain in the P site

  3. Ribosome shifts down mRNA one codon

Step 1: aminoacyl-tRNA binds to the A site (acceptor site)

Step 2: peptide bond formed between the new amino acid and the growing peptide chain in the P site

Step 3: Ribosome shifts down mRNA one codon

Translation Termination

• Stop codon on mRNA enters A site (no tRNA)

• Protein releasing factor enters the A site

• Peptidyltransferase is activated and releases the complete protein

• Ribosome recycling factor and EF-G enter the A site and the ribosome falls apart