Bacterial Transcription and Translation

Overview of Genetic Information Flow

Cells access genetic information stored in their genome through two fundamental processes: transcription and translation. These processes enable the conversion of DNA-encoded instructions into functional proteins.

• Transcription: Reading a DNA template to synthesize an RNA copy.

• Translation: Decoding the RNA to assemble proteins.

RNA Polymerases and Sigma Factors

RNA polymerase is a multi-subunit enzyme responsible for transcription in bacteria. It synthesizes RNA transcripts from a DNA template strand.

• Core polymerase: Composed of α2, β, and β' subunits; required for the elongation phase.

• Sigma factor: Required for the initiation phase; helps the core enzyme recognize the promoter, signaling the start of a gene.

• Each cell has a "housekeeping" sigma factor (e.g., Escherichia coli uses sigma-70; Bacillus subtilis uses sigma-A).

• Sigma factors recognize consensus sequences at the -10 and -35 positions relative to the RNA transcript start site (+1).

• Bacterial species can produce multiple sigma factors, allowing regulation of different sets of genes.

• Mutations in consensus sequences can alter promoter strength, affecting transcription rates ("down mutations" decrease, "up mutations" increase transcription).

Phases of Transcription

Transcription in bacteria occurs in three distinct phases:

1. Initiation: RNA polymerase holoenzyme binds to the promoter, melts the DNA helix, and synthesizes the first RNA nucleotide.

2. Elongation: Sequential addition of ribonucleotides; RNA polymerase synthesizes RNA at approximately 45 bases per second. DNA unwinding ahead of the enzyme forms a 17-base pair transcription bubble. DNA topoisomerases remove positive supercoils.

3. Termination: RNA polymerase detaches from DNA after the transcript is completed. Termination occurs via two mechanisms:

   • Rho-dependent termination

   • Rho-independent termination

Classes of RNA in Bacteria

Bacterial cells produce several classes of RNA, each serving a unique function:

• Messenger RNA (mRNA): Encodes proteins.

• Ribosomal RNA (rRNA): Forms ribosomes.

• Transfer RNA (tRNA): Shuttles amino acids to the ribosome.

• Small RNA (sRNA): Regulates transcription or translation.

• tmRNA: Frees ribosomes stuck on damaged mRNA.

• Catalytic RNA: Carries out enzymatic reactions.

RNA Stability and Degradation

RNA stability is measured by half-life. In E. coli, the average mRNA half-life is 1–3 minutes. Different RNA types have varying stabilities. The RNA degradosome is a multi-enzyme complex responsible for RNA degradation, composed of RNase, RNA helicase, and metabolic enzymes.

Translation: Decoding mRNA to Protein

Translation is the process of decoding mRNA to synthesize proteins. Ribosomes read the mRNA sequence and assemble amino acids into polypeptides.

• mRNA contains nucleotide triplets called codons, each specifying an amino acid.

• The genetic code consists of 64 codons: 61 specify amino acids (including start codons), and 3 are stop codons.

• The code is degenerate (redundant): multiple codons can encode the same amino acid.

• The genetic code is nearly universal across species.

tRNA Structure and Function

tRNAs are adaptor molecules shaped like a cloverleaf (2D) and a boomerang (3D). Each tRNA has two functional regions:

• Anticodon: Hydrogen bonds with the mRNA codon specifying an amino acid.

• 3' (acceptor) end: Binds the corresponding amino acid.

tRNAs are charged with amino acids by aminoacyl-tRNA synthetases, which recognize their specific tRNA and attach the correct amino acid.

The Ribosome: Structure and Function

Ribosomes are composed of two subunits (30S and 50S in prokaryotes), forming the 70S ribosome. Each subunit contains rRNA and proteins. The ribosome has three binding sites for tRNA:

• A (acceptor) site: Binds incoming aminoacyl-tRNA.

• P (peptidyl-tRNA) site: Holds the tRNA with the growing polypeptide chain.

• E (exit) site: Binds tRNA that has released its polypeptide.

The ribosome is a ribozyme—its peptidyltransferase activity (part of 23S rRNA) catalyzes peptide bond formation.

Reading Frame and Initiation of Translation

Ribosomes identify the correct reading frame using the Shine-Dalgarno sequence (5'-AGGAGGU-3'), a purine-rich region located 4–8 bases upstream of the start codon in E. coli. This sequence is complementary to the 3' end of 16S rRNA in the 30S subunit, facilitating ribosome binding and accurate translation initiation.

Stages of Protein Synthesis

Polypeptide synthesis occurs in three stages:

1. Initiation: Ribosomal subunits assemble, positioning the first amino acid.

2. Elongation: Amino acids are sequentially added as directed by the mRNA.

3. Termination: The completed protein is released, and ribosomal subunits are recycled.

Each stage requires protein factors and energy in the form of GTP.

Coupled Transcription and Translation

In bacteria, transcription and translation are coupled: ribosomes begin translating mRNA while it is still being synthesized by RNA polymerase. In contrast, eukaryotic microbes compartmentalize these processes.

Key Questions for Review

• How does DNA get turned into protein?

• What is a consensus sequence?

• How does sigma interact with a consensus sequence?

• How is transcription terminated?

• What is the genetic code?

• How do tRNAs become associated with the correct amino acid?

• What is the structure and function of tRNA?

• How does the ribosome know where to start translation?

• How does the ribosome identify the correct reading frame?

• What is the promoter sequence and its role in protein production?

• How does the ribosome binding site relate to protein synthesis?

• What is coupled transcription and translation?

Additional info:

• Consensus sequences are short stretches of DNA with conserved nucleotides, recognized by sigma factors to initiate transcription.

• Peptidyltransferase activity is a hallmark of ribozymes, demonstrating the catalytic potential of RNA molecules.

• Polycistronic mRNA allows multiple ribosomes to translate different proteins from a single transcript in bacteria.