Transcription, Translation, and Protein Processing

Chapter Overview

• RNA polymerases and sigma factors.
• Transcription: DNA is converted to RNA.
• The genetic code, ribosomes, and tRNAs.
• Translation: mRNA is converted to protein.
• How proteins are modified and folded.
• How proteins are degraded.
• How proteins are secreted.

Key Steps

• The cell accesses its vast store of data in its genome by:
  • Reading a DNA template to make an RNA copy (transcription).
  • Decoding the RNA to assemble protein (translation).
• After translation, each polypeptide is properly folded and placed at the correct cellular or extracellular location.

RNA Polymerases and Sigma Factors

• RNA polymerase is a complex enzyme that carries out transcription by making RNA copies (transcripts) of a DNA template strand.
• In bacteria, the RNA polymerase is made up of:
  • Core polymerase: α2, β, β'.
    • Required for the elongation phase.
  • Sigma factor: σ.
    • Required for the initiation phase.
  • Together, the core polymerase and sigma factor = holoenzyme.

Sigma Factors - Details

• The sigma factor helps the core enzyme detect the promoter, which signals the beginning of the gene.
• Every cell has a “housekeeping” sigma factor.
  • In Escherichia coli, it is sigma-70.
    • Recognizes consensus sequences at the –10 and –35 positions, relative to the start of the RNA transcript (+1).
• A single bacterial species can make several different sigma factors.

Transcription of DNA to RNA

• Transcription occurs in three phases:
  1. Initiation: RNA pol holoenzyme binds to the promoter.
     • This is followed by melting of the helix and synthesis of the first nucleotide of the RNA.
  2. Elongation: The RNA chain is extended.
  3. Termination: RNA pol detaches from the DNA, after the transcript is made.

Initiation of Transcription

• RNA polymerase holoenzyme forms a loosely bound, closed complex with DNA.
• The closed complex must become an open complex through the unwinding of one helical turn.
• RNA polymerase in the open complex becomes tightly bound to DNA and so begins transcription.
  • The first ribonucleoside triphosphate (rNTP) of the new RNA chain is usually a purine (A or G).

Transcription Elongation

• Elongation is the sequential addition of ribonucleotides from nucleoside triphosphates.
• The original RNA polymerase continues to move along the template, synthesizing RNA at ~45 bases/sec.
• The unwinding of DNA ahead of the moving complex forms a 17-bp transcription bubble.
• Positive supercoils ahead are removed by DNA topoisomerases.

Transcription Termination

• All bacterial genes use one of two known transcription termination signals:
  1. Rho-dependent
     • Relies on a protein called Rho and a strong pause site at the 3′ end of the gene.
  2. Rho-independent
     • Requires a GC-rich region of RNA, as well as 4–8 consecutive U residues.

Antibiotics That Affect Transcription

• Antibiotics must meet two fundamental criteria:
  • They must kill or retard the growth of a pathogen, and they must not harm the host.
• Rifamycin B
  • Selectively binds to the bacterial RNA pol.
  • Inhibits transcription initiation.
• Actinomycin D
  • Nonselectively binds to DNA.
  • Inhibits transcription elongation.

Different Classes of RNA = Different Functions

• Messenger RNA (mRNA): encodes proteins.
• Ribosomal RNA (rRNA): forms ribosomes.
• Transfer RNA (tRNA): shuttles amino acids.
• Small RNA (sRNA): regulates transcription or translation.
• tmRNA: frees ribosomes stuck on damaged mRNA.
• Catalytic RNA: carries out enzymatic reactions.

Transcription Comparison - Archaea and Eukarya

• Across all three domains, transcription of DNA into RNA proceeds in a similar manner.
  • Multisubunit DNA-dependent RNA-polymerases.
• Archaea and Eukaryotes differ significantly from Bacteria in the termination and initiation stages of transcription.
  • The TATA-binding protein (TBP) recognizes a motif in the promoter called the TATA box.
  • The initiator proteins remain at the promoter or are removed before elongation begins.
• Archaea and Bacteria utilize operons.
• Archaea and Eukarya RNAP exhibit homology.

Translation of RNA to Protein

• An mRNA molecule can be thought of as a sentence in which triplets of nucleotides, called codons, represent individual words, or amino acids.
• Ribosomes are the machines that read the language of mRNA and convert, or translate, it into protein via the genetic code.
• The code is degenerate or redundant.
• The code operates universally across species.
• Can turn stop codons into sense codons.
• Can introduce novel amino acids such as pyrrolysine and selenocysteine into proteins.

tRNA Molecules

• tRNAs are decoder molecules that convert the language of RNA into that of proteins.
• tRNAs are shaped like a clover leaf (in 2D) and a boomerang (in 3D).
• A tRNA molecule has two functional regions:
  • Anticodon: hydrogen bonds with the mRNA codon specifying an amino acid.
  • 3′ (acceptor) end: binds the amino acid.
• tRNAs contain a large number of unusual, modified bases.
• The charging of tRNAs is carried out by a set of enzymes called aminoacyl-tRNA synthetases.

The Ribosome Translates mRNA to Amino Acid Sequences

• Ribosomes are composed of two subunits, each of which includes rRNA and proteins.
  • In prokaryotes, the subunits are 30S and 50S and combine to form the 70S ribosome.
• The 70S ribosome harbors three binding sites for tRNA:
  1. A (acceptor) site: binds incoming aminoacyl-tRNA.
  2. P (peptidyl-tRNA) site: harbors the tRNA with the growing polypeptide chain.
  3. E (exit) site: binds a tRNA recently stripped of its polypeptide.
• The ribosome makes the peptide bonds that stitch amino acids together using peptidyltransferase.
  • Part of the 23S rRNA in the large subunit.
  • Functions as a ribozyme.
  • Functions as a molecular clock (Highly conserved, with differences in rRNA sequences that increase in relation to the evolutionary distance among species).

The Three Stages of Protein Synthesis – Require Protein Factors and GTP

• Initiation: brings the two ribosomal subunits together, placing the first amino acid in position.
  • Shine-Dalgarno sequence (finding the start): upstream, untranslated leader RNA contains a purine-rich sequence with the consensus 5′-AGGAGGU-3′.
• Elongation: sequentially adds amino acids as directed by mRNA transcript.
• Termination: releases the completed protein and recycles ribosomal subunits.

Antibiotics That Affect Translation

• Streptomycin: inhibits 70S ribosome formation.
• Tetracycline: inhibits aminoacyl-tRNA binding to the A site.
• Chloramphenicol: inhibits peptidyltransferase.
• Puromycin: triggers peptidyltransferase prematurely.
• Erythromycin: causes abortive translocation.
• Fusidic acid: prevents translocation.

Bacterial Transcription and Translation Are Coupled

• Different ribosomes can bind simultaneously to the start of each cistron within a polycistronic mRNA.
• Before RNA polymerase has even finished making an mRNA molecule, ribosomes will bind to the 5′ end of the mRNA and begin translating protein.
  • This is called coupled transcription and translation.
• Coupled transcription and translation occurs near the nucleoid.
• Translation of fully transcribed mRNA occurs at the cell poles.

Polysomes – RNA Molecule with Many Ribosomes Moving Along Its Length at the Same Time

• Ribosomes in a polysome are closely packed and arranged helically along the mRNA.
• Polysomes help protect the message from degrading RNases and enable the speedy production of protein from just a single mRNA molecule.

Protein Modification, Folding, and Degradation

• Protein typically must be modified after translation either to achieve an appropriate 3D structure or to regulate its activity.
• Protein structure may be modified after translation:
  • N-formyl group may be removed by methionine deformylase.
  • The addition of phosphoryl or methyl groups can change the activity of signal transduction.
  • The attachment of acetyl groups can regulate protein activity.
  • Lipids and sugars can be attached to proteins.
• Mass spectrometry is used to assess posttranslational modifications.
• A healthy cell “cleans house” by degrading damaged or unneeded proteins.

Protein Folding

• Folding of many proteins requires assistance from other proteins called chaperones.
  • GroEL and GroES chaperones
    • Form stacked ring with a hollow center.
    • The protein fits inside the open hole.
  • DnaK chaperones
    • Do not form rings.
    • Clamp down on a polypeptide to assist folding.

Protein Degradation: Cleaning House

• Many normal proteins contain degradation signals called degrons.
  • The N-terminal rule suggests that the N-terminal amino acid of a protein directly correlates with its stability.
• Proteasomes are protein-degrading machines found in eukaryotes and archaea.
• Bacteria contain ATP-dependent proteases, such as Lon and ClpP.

E. coli Protein Folding Versus Degradation Triage Pathways

• Damaged proteins randomly enter chaperone-based refolding pathways or degradation pathways until the protein is repaired or destroyed.

Secretion: Protein Traffic Control

• Proteins destined for the bacterial cell membrane or envelope regions require special export systems.
• Proteins meant for the cell membrane are tagged with hydrophobic N-terminal signal sequences of 15–30 amino acids and bound by the signal recognition particle (SRP) undergo cotranslational export.
• Many periplasmic proteins, such as SOD and maltose-binding protein, are delivered to the periplasm by a common pathway called the SecA-dependent general secretion pathway.
• Gram-positive bacteria must also export proteins across the cell membrane and then fold and process them once they are secreted.
• Many streptococci cluster their secretion systems at the cell membrane in an anionic phospholipid microdomain called the ExPortal.
  • The ExPortal is located near the cell septum and appears linked to peptidoglycan synthesis.

Journeys to the Outer Membrane

• Gram-negative bacteria need to export proteins completely out of the cell.
  • For example, digestive enzymes and toxins.
• Seven elegant secretion systems have evolved.
  • Labeled Type I–VII.
• ABC transporters are the simplest of the protein secretion systems and make up what is called the type I protein secretion.
• Type I protein secretion moves certain proteins directly from the cytoplasm to the environment.
• Type I systems all have three protein components:
  1. An outer membrane channel.
  2. An ABC protein at the inner membrane.
  3. A periplasmic protein lashed to the inner membrane.