BIOL303 Transcription

Thomas Cech (1981) Discovery

Discovered that RNA can serve as a biological catalyst or ribozyme, demonstrating that nucleic acids could have preceded proteins in evolution.

Ribozyme Catalyst Functions

Four capabilities: 1) Cut out their own sequence, 2) Connect RNA molecules, 3) Replicate RNA molecules, 4) Catalyze peptide bond formation.

RNA Nucleotide Structure

Consists of three parts: 1) A 5-carbon ribose sugar, 2) A phosphate group, 3) A nitrogenous base (Adenine, Guanine, Cytosine, or Uracil).

RNA vs. DNA Chemical Stability

RNA is chemically less stable and more reactive than DNA because of the 2'-hydroxyl (2'-OH) group on its ribose sugar (DNA has a hydrogen atom at the 2' position).

RNA Secondary Structure Formation

Single-stranded RNA folds into complex 3D secondary structures (like hairpins) driven by hydrogen bonding between complementary bases located on the same strand.

Ribosomal RNA (rRNA)

A class of RNA that makes up the ribosome along with ribosomal proteins; serves as the physical and catalytic site of protein assembly.

Messenger RNA (mRNA)

Carries genetic coding instructions for a polypeptide chain from DNA in the nucleus to the ribosome in the cytoplasm.

Pre-messenger RNA (pre-mRNA)

The immediate, unmodified product of transcription in eukaryotic cells; contains introns and exons and must be processed into mature mRNA.

Transfer RNA (tRNA)

An adaptor molecule that binds and brings specific amino acids to the ribosome to build a growing polypeptide chain during translation.

Small Nuclear RNA (snRNA)

Combines with small proteins to form small nuclear ribonucleoproteins (snRNPs) that participate in eukaryotic pre-mRNA processing and splicing.

Small Nucleolar RNA (snoRNA)

Participates in the chemical modification and processing of ribosomal RNA (rRNA) in eukaryotic cells.

miRNA & siRNA

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) carry out RNA interference (RNAi) to degrade target mRNA molecules or inhibit their translation.

Piwi-interacting RNA (piRNA)

Suppresses the expression and genomic movement of transposable elements (transposons) in germ cells.

Long Noncoding RNA (lncRNA)

Transcripts over 200 nucleotides long that lack start/stop codons; play key roles in regulating gene expression and chromatin structure (e.g., Xist).

CRISPR RNA (crRNA)

Found in prokaryotes; combines with Cas proteins to form a defense system that destroys foreign DNA molecules (like bacteriophages).

RNA Class Distribution (Both Domains)

RNAs transcribed in both prokaryotic and eukaryotic cells: Messenger RNA (mRNA), Ribosomal RNA (rRNA), and Transfer RNA (tRNA).

RNA Class Distribution (Eukaryote Only)

RNAs produced exclusively in eukaryotic cells: Pre-messenger RNA (pre-mRNA), Small Nuclear RNA (snRNA), Small Nucleolar RNA (snoRNA), MicroRNA (miRNA), Small Interfering RNA (siRNA), Piwi-interacting RNA (piRNA), and Long Noncoding RNA (lncRNA).

RNA Class Distribution (Prokaryote Only)

RNA produced exclusively in prokaryotes: CRISPR RNA (crRNA) used in defense.

Three Major Components of Transcription

1) A DNA template, 2) Raw materials (ribonucleoside triphosphates / rNTPs) to build the RNA, 3) Transcriptional machinery consisting of proteins/enzymes (like RNA polymerase).

Template Strand

The specific DNA nucleotide strand used for transcription; it is complementary and antiparallel to the newly synthesized RNA transcript.

Nontemplate / Coding Strand

The other DNA strand not used as a template; it is identical in sequence and direction (5' to 3') to the synthesized RNA transcript (except Uracil in RNA replaces Thymine in DNA).

Gene Transcription Direction & Strands

Different genes can be transcribed from different DNA strands; the template strand is gene-specific (e.g., Genes 'a' and 'c' are transcribed from the bottom strand, while Gene 'b' is transcribed from the top strand).

Transcription Unit

A stretch of DNA that encodes an RNA molecule and contains three critical components: 1) A Promoter, 2) An RNA-coding region, 3) A Terminator.

Promoter

A DNA sequence that the transcriptional machinery recognizes and binds to initiate transcription; determines the exact start site (+1) and direction of transcription.

RNA-coding Region

The specific DNA sequence within a transcription unit that is copied into an RNA molecule.

Terminator

A DNA sequence that signals the end of transcription and triggers the release of the RNA transcript from the RNA polymerase.

Substrates for Transcription

Ribonucleoside triphosphates (rNTPs), which consist of a ribose sugar, a nitrogenous base, and three phosphate groups.

Chemical Direction of RNA Synthesis

RNA is synthesized in the 5' to 3' direction; the template DNA strand is read in the 3' to 5' direction by the transcription machinery.

Phosphodiester Bond Formation Mechanics

The 3'-OH group of the growing RNA strand attacks the 5'-alpha-phosphate of the incoming rNTP, cleaving two phosphates (pyrophosphate, PPi) and forming a covalent phosphodiester bond.

Primer Requirement in Transcription

None! Unlike DNA polymerases, RNA polymerases do NOT require a primer to initiate the synthesis of an RNA molecule.

Bacterial RNA Polymerase

Bacteria have a single, universal RNA polymerase that catalyzes the synthesis of all classes of cellular RNA (mRNA, tRNA, rRNA).

Bacterial Core RNA Polymerase Subunits

Composed of 5 subunits: two alpha (α) subunits, one beta (β) subunit, one beta-prime (β') subunit, and one omega (ω) subunit (α2ββ'ω). It can synthesize RNA but cannot recognize promoters specifically.

Sigma (σ) Factor

A bacterial transcription factor that controls the specific binding of RNA polymerase to the promoter; dissociates after initiation.

Bacterial RNA Polymerase Holoenzyme

Formed by the association of the core RNA polymerase enzyme and the sigma (σ) factor; required for promoter recognition and initiation.

Eukaryotic RNA Polymerase I

Eukaryotic enzyme present in all eukaryotes; transcribes large ribosomal RNA (large rRNAs).

Eukaryotic RNA Polymerase II

Eukaryotic enzyme present in all eukaryotes; transcribes pre-messenger RNA (pre-mRNA), snoRNAs, miRNAs, and most snRNAs.

Eukaryotic RNA Polymerase III

Eukaryotic enzyme present in all eukaryotes; transcribes transfer RNA (tRNA), small ribosomal RNA (small rRNA), miRNAs, and some snRNAs.

Eukaryotic RNA Polymerases IV & V

Enzymes present only in plants; transcribe siRNAs involved in silencing transposable elements (IV) and altering chromatin structure (V).

Three Stages of Transcription

1) Initiation (transcription apparatus assembles on promoter and synthesis begins), 2) Elongation (polymerase unwinds DNA and adds nucleotides to the 3' end), 3) Termination (end is recognized, transcript and enzyme release).

Bacterial Transcription Initiation Steps

1) Holoenzyme recognizes promoter, 2) DNA is unwound to form a transcription bubble, 3) First rNTP is added at +1 site, 4) Transcription apparatus escapes the promoter.

Consensus Sequence

The set of the most commonly encountered nucleotides among sequences that share structural or functional similarity.

Bacterial -10 Consensus Sequence

Also called the Pribnow box; located 10 bp upstream of the start site. Its consensus sequence is TATAAT.

Bacterial -35 Consensus Sequence

Located 35 bp upstream of the start site; its consensus sequence is TTGACA.

Upstream Element (UP element)

An A-T rich consensus sequence located between -40 and -60 in some bacterial promoters; significantly enhances the rate of transcription initiation.

Bacterial Abortive Initiation

A process where bacterial RNA polymerase repeatedly synthesizes and releases short RNA transcripts (2 to 6 nucleotides in length) while remaining bound to the promoter before successfully escaping.

Promoter Escape / Clearance (Bacteria)

Occurs when bacterial RNA polymerase successfully synthesizes an RNA molecule 9 to 12 nucleotides long, undergoes a conformational change to escape the promoter, and releases the sigma factor.

Bacterial Terminator Types

1) Rho-dependent terminators (require the rho protein to stop transcription), 2) Rho-independent terminators (can terminate transcription autonomously without rho).

Polycistronic mRNA

A single bacterial mRNA transcript that contains the coding sequences for multiple, functionally related genes in an operon, allowing co-expression.

Rho-dependent Termination Mechanism

1) Rho protein (ρ) binds to a 'rut' (rho utilization) site on the newly transcribed RNA, 2) Rho moves 5' to 3' chasing RNA polymerase, 3) RNA pol pauses at a terminator sequence, 4) Rho catches up and uses helicase activity to unwind the DNA-RNA hybrid, terminating transcription.

Rho-independent Termination Mechanism

1) Transcription of inverted repeats that fold into a hairpin loop on the RNA, causing RNA polymerase to pause, 2) A sequence of ~6 adenine nucleotides on the template DNA is transcribed into a string of uracils (U's) on the RNA, 3) The weak A-U base pairing destabilizes the DNA-RNA hybrid, causing the transcript to release.

Eukaryotic Transcription Factors

Proteins that bind to specific eukaryotic DNA regulatory sequences to influence transcription rate and position RNA polymerase.

Eukaryotic Basal Transcription Apparatus

The minimal transcription complex that assembles near the transcription start site; composed of RNA polymerase II, general transcription factors (GTFs), and Mediator.

Eukaryotic Core Promoter

The region immediately upstream of the transcription start site where the basal transcription apparatus binds; contains key consensus sequences, most commonly the TATA box (consensus TATAAA) located at -25 to -35 bp.

TFIIB Recognition Element (BRE)

A eukaryotic core promoter consensus sequence located at -35 bp with the consensus sequence G/C G/C G/C CGCC.

Initiator Element (Inr)

A eukaryotic core promoter consensus sequence encompassing the transcription start site (+1) with the consensus sequence YYANT/AYY (Y = pyrimidine, N = any nucleotide).

Downstream Core-promoter Element (DPE)

A eukaryotic core promoter consensus sequence located at +30 bp downstream of the start site; consensus sequence is RG A/T CGTG (R = purine).

Eukaryotic Regulatory Promoter

Located immediately upstream of the core promoter; contains various consensus sequences where transcriptional activator proteins bind to regulate transcription frequency.

Eukaryotic Enhancers

Distant DNA sequences (either upstream, downstream, or within introns) where transcription factors bind to stimulate maximal transcription levels.

Eukaryotic Internal Promoters

Promoters located downstream of the start site within the transcribed coding sequence; typical of small rRNA and tRNA genes transcribed by RNA Polymerase III.

Eukaryotic Pre-initiation Complex (PIC)

The macromolecular assembly of RNA Polymerase II, general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH), and Mediator at the core promoter.

TFIID and TATA-binding Protein (TBP)

TFIID is the first general transcription factor to bind the core promoter; it contains TBP, which binds to the minor groove of the TATA box, bending and unwinding the DNA helix.

Mediator Complex in Eukaryotes

A multi-protein complex that serves as a physical bridge between transcriptional activator proteins bound to regulatory promoters or enhancers and the basal transcription apparatus.

Eukaryotic DNA Looping

Allows transcription factors bound to distant enhancers to physically interact with the basal transcription apparatus at the promoter by bending the intervening DNA.

Eukaryotic Transcription Elongation & Clearance

After synthesizing ~30 bp of RNA, RNA Polymerase II undergoes a conformational change, sheds most general transcription factors, escapes the promoter, and continues elongation 5' to 3' in the transcription bubble.

Eukaryotic RNA Polymerase I Termination

Requires a specific protein termination factor that binds to a specific DNA sequence downstream of the transcription unit (similar to Rho, but binds DNA rather than RNA).

Eukaryotic RNA Polymerase II Termination Mechanism

Occurs via cleavage and the Rat1 exonuclease pathway. 1) RNA Pol II transcribes past what is needed, 2) The RNA is cleaved at a 3' coding site, releasing mature mRNA, 3) Yeast 5'->3' exonuclease Rat1 binds to the 5' end of the trailing RNA, chews it up, and catches the polymerase to stop transcription.

Eukaryotic RNA Polymerase III Termination

Terminates transcription autonomously after transcribing a poly-uracil (poly-U) terminator sequence and upstream secondary structures (hairpins), requiring no additional proteins.