RNA Transcription and Gene Expression

• RNA transcription and the control of gene expression are vital for expressing DNA information as ribonucleic acid (RNA)
• This is a tightly regulated process
• A fundamental biologic principle known as the central dogma describes it.
• The three major classes of RNA produced are those that contain the information needed to synthesize a polypeptide and those with structural and catalytic functions for polypeptide synthesis

RNA Classification

• Messenger RNA (mRNA) serves as the blueprint for protein synthesis but is the least abundant and most heterogeneous RNA species
• Eukaryotic mRNA features a 7-methylguanosine cap and a polyA tail post-synthesis
• Ribosomal RNA (rRNA) associates with ribosomal proteins to form ribosomes, the cellular workbenches for polypeptide assembly; it makes up to 80% of total RNA
• Prokaryotes possess three rRNA sizes: 23S, 16S, and 5S
• Eukaryotes have four: 28S, 18S, 5.8S, and 5S ("S" denotes Svedberg unit for molecular size)
• Transfer RNA (tRNA) is the smallest RNA type (around 4S)
• There is at least one unique tRNA for each of the 20 amino acids and it has tertiary structure with unique bases like pseudouracil and dihydrouracil

RNA Transcription

• RNA is transcribed from a DNA template by RNA polymerase
• Prokaryotic cells utilize one multisubunit RNA polymerase for transcribing all three major RNA classes
• Eukaryotic cells have specialized RNA polymerases

RNA Polymerase specifics

• RNA polymerase I makes rRNA.
• RNA polymerase II makes mRNA.
• RNA polymerase III makes tRNA, 5S RNA, and other small RNAs

Prokaryotic Transcription of mRNA

• RNA synthesis from a DNA template is transcription, but not all gene DNA is transcribed
• Genes feature regulatory promoter sequences and transcription units
• In prokaryotes, the transcription unit has a 5' promoter and 3' termination signal
• The coding strand is the sense strand, and the opposite is the template or antisense strand
• Untranslated regions (UTRs) on both ends of the coding region are on the mRNA and serve functions during polypeptide synthesis (translation)
• The prokaryotic 5' UTR contains a Shine-Dalgarno sequence for mRNA binding to the ribosome
• Prokaryotic RNA polymerase has four subunits, known as the core enzyme, with 5' to 3' polymerase activity
• Two associated proteins help it bind correctly, transcribe DNA, and terminate the process
• The σ (sigma) factor, a dissociable protein, is required for transcription initiation
• Together with the core enzyme, it forms the holoenzyme
• The p (rho) factor is required for chain termination in some cases
• The first step in transcription initiation is binding RNA polymerase holoenzyme to the promoter region
• The promoter contains two sites that bind and position RNA polymerase: the -10 site (Pribnow box) and the -35 site
• The Pribnow box, rich in AT pairs, melts easily and allows RNA polymerase to open the helix
• Rifamycin antibiotics inhibit transcription initiation by inactivating RNA polymerase
• During elongation, RNA polymerase unwinds and rewinds the DNA helix and almost two full turns of DNA creates a transcription bubble
• Supercoiling strain is created ahead of the helix, but the strain is relieved when the DNA helix rewinds at the other end of the bubble
• RNA chain elongation is achieved by adding 5' nucleoside triphosphates to the 3' hydroxyl of the growing RNA chain
• Transcription is always in the 5' to 3' direction, similar to DNA polymerization
• RNA polymerase does not proofread the RNA sequence
• Either strand of DNA can serve as the template, but synthesis always runs 5' to 3', thus the template DNA strand is always read 3' to 5'
• The antibiotic actinomycin D inhibits RNA polymerase by binding strongly to the DNA helical structure
• The prokaryotic chain termination signal is a hairpin structure and has two classes, which either contain a string of T's after the hairpin structure on the coding strand, or lack the string and require the p protein

Eukaryotic Transcription of mRNA:

• Eukaryotic genes have some similarities to prokaryotic genes
• The transcription unit has a promoter on the 5' end and a termination signal on the 3' end and the coding region is flanked by UTRs.
• There is no Shine-Dalgarno sequence
• The eukaryotic version is divided into segments (exons and introns)
• Exons (expressed sequences) correspond to functional polypeptide segments called domains
• Introns (intervening sequences) do not code for polypeptide structure
• The eukaryotic promoter is more complex with a TATA box and CAAT box located upstream of the start site
• For genes transcribed by RNA polymerase II (for mRNA synthesis), the basal transcription complex forms on a short DNA sequence known as the TATA box
• RNA polymerase II binds to transcription factor (TFII) to initiate transcription
• Once an active transcription complex is produced, then RNA synthesis proceeds as in prokaryotes

Processing of Primary RNA Transcripts:

• For prokaryotes the primary mRNA transcript is functional immediately, in eukaryotes
• The RNA transcript must undergo prior processing in the nucleus, which involves 5' capping, 3' polyadenylation (polyA tailing), and exon splicing
• Capping involves adding an inverted 7-methylguanosine triphosphate to the 5' end to produce a 5' to 5' phosphodiester bond, therefore providing a free 3' hydroxyl at the 5' end of the molecule, and the polyA tail is attached at the 3' end by polyA polymerase
• Capping and polyadenylation stabilize the mRNA by blocking access to the termini by exonucleases, and allowing them to participate in polypeptide chain initiation
• Splicing removes introns via splice sites or splice junctions
• Splice junctions start with a consensus sequence (GU) and end with AG
• Specialized small nuclear RNA particles recognize the donor and acceptor sites and associate with nuclear proteins to form spliceosomes
• Primary ribosomal and transfer RNA transcripts need to be processed by nucleases

Transcriptional Control of Gene Expression

• Individual cell types must regulate the expression of necessary genes because all cells in the body have the same genetic composition
• Gene expression is controlled with transcriptional regulation
• The lack of a nuclear membrane in prokaryotes allows ribosomes to access mRNA transcripts and is a major point of regulation

Regulation of the Lac Operon

• The lac operon is a genetic unit that produces lactose digestion enzymes and consists of three contiguous structural genes transcribed as continuous mRNA by RNA polymerase
• An operator sequence at the 5' end serves as a binding site for a repressor protein that blocks RNA polymerase, which is produced constitutively by the i gene
• The inducer, allolactose, binds to the repressor subunits, preventing their assembly into an active tetramer
• Another regulatory component is the catabolite activator protein (CAP). CAP forms an active complex with intracellular cyclic adenosine monophosphate (cAMP), and binds with RNA polymerase to allow the lac operon to be expressed when glucose is absent
• Lac operons exhibit both negative and positive control
• Conditions: glucose only; prevents expression of lac operon if both lactose and glucose are present
• Positive control (Conditions: Lactose only; permits expression of lac operon).

Eukaryotic Transcriptional Control

• Achieved through controlling the physical accessibility of DNA to RNA polymerase or regulating the rate of RNA polymerase binding to the promoter
• Condensed chromatin, known as heterochromatin, has inactive genes and is highly methylated mostly in cytosine residues in CpG islands
• Euchromatin is highly acetylated
• RNA polymerase binding to the promoter is controlled by binding transcription proteins to certain DNA sequences
• A cis-acting element includes the CAAT box, the TATA box, and the GC box, and can include enhancer or silencer sequences
• Most transcription factors have three domains:
  • DNA binding.
  • Activation
  • Dimerization

Gene Amplification

• A gene amplification refers to an increase in the copies of the same gene
• Examples of gene amplification are the ribosomal genes and histone genes that are tandem
• It is required in embryonic development and in differentiating tissues (such as embryonic development) where a large amount is needed for specific processes

Alternative Splicing

• Alternative splicing is a mechanism for generating multiple protein species from an RNA transcript of a single gene
• This includes alternative tailing.

Messenger RNA Editing

• The coding information in a gene can be changed after processing of the primary transcript by RNA editing
• The best known example is the editing of the mRNA for apolipoprotein B (apoB)

RNA Interference and “Gene Silencing”

• A promising new technology used for human therapy and based on a discovered mechanism for blocking RNA translation via RNA interference
• There are RNA interference components ubiquitous in eukaryotes and are a "dicer" protein and an RNA-induced silencing complex.
• This includes using small hairpin RNAs, also known as microRNAs, as a template to recognize and block the translation of mRNA with reverse complementary sequences.