Transcription and RNA: Chapter 13

• Chapter 13 covers the process of transcription

RNA and Cellular Functions

• Evidence suggests RNA was the original genetic material
• Ribozymes are catalytic RNA molecules

RNA Structure

• RNA includes a primary structure of a single strand
• RNA includes a secondary structure formed through folding

Differences Between DNA and RNA Structures

• DNA has deoxyribose as its sugar while RNA contains ribose
• DNA does not have a 2'-OH group, RNA does
• DNA uses the bases A, G, C, and T, while RNA uses A, G, C, and U
• DNA has a double helix secondary structure; RNA has many types of secondary structure
• DNA is stable, whereas RNA is easily degraded

Classes of RNA

• Ribosomal RNA (rRNA) are structural and functional components of the ribosome
• Messenger RNA (mRNA) carry genetic codes for proteins
• Transfer RNA (tRNA) helps incorporate amino acids into polypeptide chains
• Small nuclear RNAs (snRNAs) are involved in processing pre-mRNA
• Small nucleolar RNAs (snoRNAs) process and assemble rRNA
• MicroRNAs (miRNAs) inhibit mRNA translation
• Small interfering RNAs (siRNAs) trigger degradation of RNA molecules
• Piwi-interacting RNAs (piRNAs) suppress the transcription of transposable elements in reproductive cells
• CRISPR RNA (crRNA) assists in the destruction of foreign DNA (prokaryotes)
• Long noncoding RNA (lncRNA) impacts different functions (eukaryotes)

Transcription Overview

• Transcription necessitates a DNA template; raw materials (ribonucleotide triphosphates); and the transcription apparatus
• The transcribed strand serves as the template strand
• The transcription unit includes a promoter, an RNA-coding sequence, and a terminator
• Under an electron microscope, DNA molecules undergoing transcription look like Christmas trees
• RNA molecules are synthesized complementary and antiparallel to the template strand

Template Strand

• The template strand is the DNA strand copied into an RNA molecule
• Genes can be transcribed from either DNA strand

Transcription Unit

• The transcription unit has a promoter, an RNA-coding region, and a terminator

Transcription Substrates

• Ribonucleoside triphosphates (rNTPs) are added to the 3' end of the RNA molecule
• Initial RNA synthesis does not require a primer
• Nucleotides are always added to the 3' end of the RNA molecule

Transcription Apparatus

• Bacterial RNA polymerase includes five subunits of the core enzyme; two copies of a, single copies of B and B', and a stabilizing enzyme w
• The sigma (σ) factor binds to the promoter to initiate transcription in bacteria

Eukaryotic RNA Polymerases

Bacterial Promoters

• Bacterial promoters possess consensus sequences, which are sequences with significant similarities
• The -10 consensus sequence (Pribnow box) is 10 bp upstream of the start site: 5' TATAAT 3' and 3' ATATTA 5'
• The -35 consensus sequence is TTGACA and is found upstream from the start site

Transcription Initiation

• Sigma factor associates with the core enzyme to form a holoenzyme
• The holoenzyme binds to the -35 and -10 consensus sequences in the promoter
• The holoenzyme binds the promoter tightly and unwinds the double-stranded DNA
• An rNTP bases connect at the start site and serves as the first nucleotide in the RNA molecule
• Two phosphate groups are cleaved from each subsequent rNTP, creating an RNA nucleotide that is added to the 3' end of the growing RNA molecule
• Sigma factor releases as the RNA polymerase moves beyond the promoter to initiate transcription

Transcription Elongation

• RNA elongation is carried out by the action of RNA polymerase

Transcription Termination

• Rho-dependent termination uses the rho factor
• Rho-independent termination uses a hairpin structure formed by inverted repeats, followed by a string of uracils

Rho-Dependent Termination Summary

• The rho factor binds to the rut site and travel toward the 3’ end
• Once RNA polymerase encounters a terminator sequence, it pauses, allowing rho to catch up.
• Using helicase activity, rho unwinds the DNA-RNA hybrid and brings an end to transcription

Rho-Independent Termination Summary

• An inverted repeat is transcribed into RNA
• The string of Us causes the RNA polymerase to pause
• Inverted repeats in RNA fold into a hairpin
• Hairpin destabilizes the DNA-RNA pairing causing temination.

Eukaryotic Transcription

• Eukaryotic transcription has some differences with bacterial transcription
• Eukaryotic transcription requires chromatin modification before transcription
• Core promoter usually has a TATA box; TATAAAA, -25 to -30 bp, is bound by transcription factors

Promoters

• TFIIB recognition element
• Initiator element
• Downstream core promoter element

Transcription Initiation

• TFIID binds to TATA box through the TATA binding protein (TBP) in the core promoter
• The holoenzyme contains general transcription factors, RNA polymerase II, and the mediator

Eukaryotic Transcription Elongation

• The DNA double helix enters RNA polymerase II through a cleft in the enzyme and unwinds
• The DNA-RNA duplex is bent at a right angle, positioning the 3' end of the RNA at the active site of the enzyme
• New nucleotides are added to the 3' end of the growing RNA molecule

Eukaryotic Termination

• Eukaryotic termination involves exonuclease enzymes
• The exonuclease enzyme attaches to the cleaved 5' end of the RNA
• The exonuclease enzyme moves down the RNA and degrades it until transcription is terminated when it reches the RNA polymerase

Transcription in Archaea

• Transcription in Archaea is similiar to Transcription in Eukaryotes
• It suggests a closer relationship between archaea and eukaryotes
• Archaea possess RNA polymerase, promoters, and TATA-binding proteins like eukaryotes