Transcription Quiz

• Transcription is RNA synthesis using DNA strands as templates, transferring genetic information from DNA to RNA.

Central Dogma: DNA to RNA to Protein

• The central dogma outlines the flow of genetic information: DNA undergoes replication and transcription into RNA.

• RNA is then translated into proteins.

• Reverse transcription is the process where RNA is converted back to DNA.

RNA Types and Functions

• mRNAs (messenger RNAs) code for proteins.
• rRNAs (ribosomal RNAs) form ribosomes and catalyze protein synthesis.
• tRNAs (transfer RNAs) act as adaptors between mRNA and amino acids.
• Telomerase RNA serves as a template for chromosome end extension.
• snRNAs (small nuclear RNAs) function in nuclear processes like pre-mRNA splicing.
• snoRNAs (small nucleolar RNAs) process and modify rRNAs.
• lncRNAs (long noncoding RNAs) regulate cell processes like X-chromosome inactivation.
• miRNAs (microRNAs) regulate gene expression by blocking mRNA translation or causing degradation.
• siRNAs (small interfering RNAs) turn off gene expression by degrading mRNAs or establishing repressive chromatin structures.
• piRNAs (Piwi-interacting RNAs) protect the germ line from transposable elements.

Template and Coding Strands

• The template strand is used by RNA polymerase to create RNA.
• The coding strand has a sequence identical to the RNA, except with Thymine instead of Uracil.

Structural Genes

• DNA regions that can be transcribed into RNA include promoters and structural genes.

RNA Polymerase

• RNA polymerase is responsible for RNA synthesis.
• In E. coli, it consists of subunits α2ββ'ωσ.
• Subunits determine the DNA to be transcribed, catalyze polymerization, bind and open the DNA template, and recognize the promoter.

Rifampicin

• Rifampicin, used for tuberculosis, inhibits RNA synthesis by binding to the RNA polymerase’s β subunit and acting as a polymerase inhibitor.

Operons

• Transcriptable regions of DNA are called transcription units.

• In E. coli one unit includes both promoter and operator genes.

• An operon includes structural genes and upstream DNA containing regulatory sequences

Promoters

• Promoters are DNA sequences where RNA polymerase binds to initiate transcription.

Transcription Process in Prokaryotes

• Initiation Phase: RNA polymerase recognizes the promoter and starts transcription.
• Elongation Phase: The RNA strand grows continuously.
• Termination Phase: RNA polymerase stops synthesis, and the RNA separates from the DNA.

Initiation in Prokaryotes

• The first nucleotide on an RNA transcript is always a purine triphosphate.
• GTP is more common than ATP.

Elongation in Prokaryotes

• Free nucleotide triphosphates add sequentially to the 3'OH end of the nascent RNA strand.

Termination in Prokaryotes

• The newly synthesized RNA and RNA polymerase separate.
• Termination can be intrinsic (sequences) or rho-dependent (proteins).

Rho-dependent Termination

• Rho-dependent termination requires rho factor, a protein.
• The rho factor binds to the RNA and moves along it to interact with RNA polymerase, releasing it.
• The rho factor is a hexameric, ATP-dependent RNA translocase that terminates transcription at rho utilization sites.

Rho-independent Termination

• Termination is determined by specific sequences (termination sites).
• Termination sites consist of inverted repeats, rich in G:C that form a stem-loop in RNA.
• A series of 7 U residues following the hairpin stem causes RNA polymerase to pause.
• The U-rich sequence is unable to hold the RNA-DNA hybrid together leading to termination.

Transcription in Eukaryotes

• In eukaryotes, the transcription takes place in the nucleus.
• In bacteria, the transcription and translation take place simultaneously.

RNA Polymerases in Eukaryotes

• RNA polymerase I transcribes 5.8S, 18S, and 28S rRNA genes.
• RNA polymerase II transcribes protein-coding genes, snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, and most snRNA genes.
• RNA polymerase III transcribes tRNA genes, 5S rRNA genes, some snRNA genes, and genes for other small RNAs.
• RNA polymerase I is not sensitive to Amanitin.
• RNA polymerase II is highly sensitive to Amanitin.
• RNA polymerase III has moderate sensitivity to Amanitin.
• Amanitin specifically inhibits RNA polymerase.

Cis-Acting Elements

• Cis-acting elements are DNA sequences that regulate transcription of genes on the same DNA molecule.
• A Hogness box, also known as a TATA box, is a DNA sequence found in the promoter regions of genes in eukaryotes and archaea.

Pre-Initiation Complex (PIC)

• TBP (TATA binding proteins) of TFII D binds to TATA box in the promoter region.
• TFII A and TFII B then bind to TFII D
• TFII F-RNA-pol complex binds to the already bound TFII B.
• TFII then facilitates opening of dsDNA (helicase and ATPase activity)
• Finalized by completion of PIC (preinitiation complex) with additional factors.

Eukaryotic Transcription Factors

• TFIID recognizes the TATA box.
• TFIIB recognizes the promoter and positions RNA polymerase.
• TFIIA stabilizes TFIID binding.
• TFIIF stabilizes RNA polymerase interaction with TFIIB.
• TFIIE attracts and regulates TFIIH.
• TFIIH unwinds DNA and phosphorylates the RNA polymerase C-terminal domain.

Activators

• Gene regulatory proteins help RNA polymerase the general factors assemble at the promoter.

Mediators

• Mediate between activator proteins and Pol II and General Transcription Factors.

Elongation

• Stabilize binding of RNA polymerase molecule.
• Transcription and translation do not take place simultaneously separated by nuclear membrane.

DNA Topoisomerase and Gyrase

• DNA topoisomerase rapidly removes superhelical tension in DNA.
• DNA gyrase, uses ATP hydrolysis to continuously pump supercoils into the gyrase thereby maintaining DNA under constant tension.

Eukaryotic Termination

• The common eukaryotic termination sequence is AATAAA followed by GT repeats.
• Termination may be Rho factor dependent or independent.

Modification of hnRNA

• Post-transcriptional modifications include:
  • Capping at the 5' end, important for recognition by ribosomes and protection from degradation.
  • Tailing (Polyadenylation) at the 3' end which increases mRNA stability.
  • mRNA splicing (introns removal).

Splicing

• Primary transcripts of mRNA are called as heteronuclear RNA (hnRNA).
• The capping occurs before the splicing.
• The cap structure of messenger RNA is recognized by the cap-binding protein required for translation.

Split Genes, Exons, and Introns

• Split genes consist of both introns and exons, where exons are coding and introns aren't.
• Exons are the coding sequences in split genes, appearing on both genes and primary transcripts, and expressed to mature mRNA.
• Introns are non-coding sequences in primary mRNA transcripts that are removed during splicing.

Spliceosome

• The spliceosome performs pre-mRNA splicing, composed of small nuclear RNAs (snRNAs) and proteins.

RNA Editing

• RNA editing involves changes (insertion, deletion, base substitution) to specific nucleotide sequences within an RNA molecule after it has been generated.
• The changes affect multiple functions of a gene, including localization and stability of RNAs.

Prokaryotic vs. Eukaryotic Transcription

• In prokaryotes, mRNA is transcribed, immediately translated without modification.
• In eukaryotes, mRNA undergoes several post-transcriptional modifications like 5' methyl capping and poly A tail and splicing the nucleoplasm

Test your understanding of transcription, the process of synthesizing RNA from a DNA template. Questions cover template usage, RNA sequencing, and the role of RNA polymerase in eukaryotic cells.