Bacterial Transcription and RNA Synthesis

RNA Structure & Synthesis (I)

• RNA has three main types crucial for protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA).
• Ribosomal RNA (rRNA) is associated with several proteins in ribosomes, serving as protein synthesis sites. Prokaryotic rRNA includes 23S, 16S, and 5S; eukaryotic rRNA includes 28S, 18S, 5.8S, and 5S. The S values (Svedberg units) relate to molecular weight and shape. rRNA accounts for approximately 80% of total RNA in a cell.
• Transfer RNA (tRNA) is the smallest RNA molecule (4S), with at least one specific type for each of the 20 amino acids commonly found in proteins. It comprises about 15% of total RNA. tRNA acts as an adaptor, carrying its specific amino acid covalently attached to its 3' end to protein synthesis sites. It recognizes the genetic code word on mRNA, determining which amino acid is added to the growing peptide chain.
• Messenger RNA (mRNA) makes up only about 5% of RNA. It is the most heterogeneous RNA type, varying in size (500 to 6000 nucleotides) and base sequence. mRNA carries genetic information from nuclear DNA to the cytosol. mRNA can be polycistronic (carrying information from multiple genes, characteristic of prokaryotes) or monocistronic (carrying information from a single gene, characteristic of eukaryotes). Eukaryotic mRNA has a poly-A tail (long sequence of adenine nucleotides) at the 3' end and a "cap" (molecule of 7-methylguanosine) at the 5' end.

Prokaryotic and Eukaryotic rRNAs

• Prokaryotes have 16S, 23S, and 5S rRNA.
• Eukaryotes have 18S, 5.8S, 28S, and 5S rRNA.

Prokaryotic RNA Polymerase

• RNA polymerase in bacteria synthesizes all RNA except for short RNA primers required for DNA replication.
• It's a multisubunit enzyme recognizing a nucleotide sequence (the promoter region) at the beginning of a transcribed DNA segment.
• It creates a complementary RNA copy from the DNA template strand.
• It recognizes the end (terminal region) of the DNA sequence to be transcribed.

Core Enzyme

• The core enzyme is required for enzyme assembly.
• It's responsible for template binding and 5'→3' RNA polymerase activity.
• Critically, it cannot recognize the promoter region on DNA.

Holoenzyme

• The addition of the σ subunit (sigma factor) creates the holoenzyme.
• This enables RNA polymerase to recognize promoter regions on DNA.
• Different σ factors recognize different gene groups.

Termination Factor

• Some DNA regions signal transcription termination directly recognized by the RNA polymerase itself.
• Other termination sequences are recognized by specific termination factors (like rho (ρ) factor in E. coli).

Steps in RNA Synthesis (Prokaryotes)

• RNA synthesis occurs in three phases: initiation, elongation, and termination.
• Initiation: RNA polymerase holoenzyme binds to the promoter region. Prokaryotic promoters have characteristic consensus sequences like -35 and Pribnow (or -10) box. The -35 sequence (5'-TTGACA-3') is centered about 35 nucleotides from the transcription start point and the Pribnow box (5'-TATAAT-3') is centered about -10.
• Elongation : Holoenzyme recognizes the promoter region and starts synthesizing DNA sequence (often a purine) transcript. Elongation phase starts when the transcript exceeds ten nucleotides. The σ subunit is released during elongation.

Elongation (cont'd...)

• RNA polymerase doesn't use a primer.
• It doesn't have proofreading activity.
• It uses ribonucleoside triphosphates and releases pyrophosphates during nucleotide addition to growing chains.
• Binding of RNA polymerase to the DNA template causes a local unwinding of the DNA helix, leading to the formation of supercoils that can be relaxed by DNA topoisomerase.

Termination

• The RNA chain elongation process continues until a termination signal is reached.
• Depending on the signal, either p-dependent termination (requiring an additional Rho (ρ) factor) or p-independent termination (no additional factor needed). P-independent termination is characterized by the presence of a hairpin loop in the nascent RNA, which helps halt transcription.

Action of Antibiotics

• Some antibiotics inhibit bacterial RNA synthesis, and this can prevent bacterial growth.
• Rifampin is an example, binding to the bacterial RNA polymerase subunit (β), hindering transcription initiation and the first phosphodiester bond formation.

Transcription from bacterial operons

• In bacteria, structural genes for metabolic pathway enzymes often group on chromosomes along with regulatory genes, forming transcription units called operons.
• These genes are coordinately expressed as a long mRNA molecule. The lactose operon in E. coli is a key example.

The Lactose (lac) Operon

• The lac operon controls three enzymes involved in lactose catabolism: β-galactosidase (lacZ), permease (lacY), and thiogalactoside transacetylase (lacA).
• The regulatory portion of the operon involves a catabolite activator protein (CAP or CRP) binding site, the promoter, and the operator site.

Lactose Operon (conditions)

• Off (with glucose): The repressor protein binds to the operator site, blocking RNA polymerase. No mRNA, and therefore, no proteins are produced.

• On (with lactose): Lactose (converted to allolactose) binds to the repressor protein, causing a conformation change that prevents it from binding to the operator site. RNA polymerase can initiate transcription to produce mRNA, which is then translated into proteins necessary for lactose metabolism

• Operon with glucose and lactose (off): When both glucose and lactose are present, adenyl cyclase is deactivated, preventing cAMP production. A cAMP-CAP complex can't form, and the CAP binding site remains empty. Even though the repressor is unbound, RNA polymerase still cannot effectively initiate transcription, so the lactose genes are not expressed.