Introduction

• Gene: an organized unit of DNA sequences that enables a segment of DNA to be transcribed into RNA and ultimately results in the formation of a functional product

  • Other genes code for RNA itself as a product

    • Transfer RNA (tRNA): translates mRNA into amino acids

    • Ribosomal RNA (rRNA): part of ribosomes

• Eukaryotes also have an intervening step called RNA processing

  • RNA processing: pre-mRNA is processed into active mRNA

• Some genes do not encode polypeptides

  • An RNA is the final functional product

    • Structural RNAs

    • Regulatory RNAs

ncRNA Functions

• Scaffold: bind to multiple components such as proteins, act as scaffold for formation of a complex

• Guide: guide one molecule to a specific location in the cell

• Alteration of protein function or stability: binds to a protein, can affect ability of the protein to be a catalyst, ability of the protein to bind other molecules, protein stability

• Ribozymes: RNA molecule with catalytic function

• Blocker: prevents or blocks a cellular process from happening

• Decoy: recognize other ncRNAs and sequester them, preventing them from working

Differences in Prokaryotic and Eukaryotic Transcription

• Prokaryotes

  • All RNA species are synthesized by a single RNA polymerase

  • mRNA is translated during transcription

  • Genes are contiguous segments of DNA that are colinear with the mRNA that is translated into a protein.

  • mRNAs are often polycistronic

• Eukaryotes

  • 3 different RNA polymerases for the different classes of RNA molecules.

  • mRNA is processed before transport to the cytoplasm. Caps and tails are added and introns are removed.

  • Genes are often split. Exons are separated by introns

  • mRNAs are monocistronic

RNA Polymerase (RNAP)

• Synthesizes RNA in a 5’🡪 3’ direction

• Requires a DNA template (read in a 3’ to 5’ direction)

• Are large complexes of proteins

• Prokaryotic: single type of RNAP

  • Contains four catalytic subunits and a single regulatory subunit known as sigma (s).

    • α2ββ’

  • Distinct sigma factors

    • Are sequence specific DNA binding proteins

    • Reduce non-specific DNA binding of the RNAP

    • Increase specific binding to the promoters

• Eukaryotic RNA polymerases have different roles in transcription

  • Polymerase I: makes a large precursor to the major rRNA (5.8S,18S and 28S rRNA in vertebrates)

  • Polymerase II: synthesizes hnRNAs, which are precursors to mRNAs. It also make most small nuclear RNAs (snRNAs)

  • Polymerase III: makes the precursor to 5SrRNA, the tRNAs and several other small cellular

    and viral RNAs.

Promoters

• Promote assembly of the RNA pre-initiation complex

• Position the transcription start site

• Control the directionality of transcription

• Location and orientation of promoter dictates which strand of dna is used for transcription

Directions of Transcription

• Direction of transcription and which DNA strand used varies among genes

• In all cases, synthesis of RNA transcript is 5’ to 3’ and DNA template strand reads 3’ to 5’

• Antisense strand: used a a template

• Sense strand: not the template strand and has the same sequence as the RNA molecule

Promoter Sequences

• Prokaryotes

  • A sequence of TATAAT centered 10 nucleotides upstream of the +1 site and is called the Pribnow box

  • The sequence TTGACA centered at position -35 and is the site of sigma factor binding

• Eukaryotes

  • TATA box located about 25 nucleotides upstream from the transcriptional start site.

  • CAAT box at position  -70

Eukaryotic Promoters

• The basal or core promoter is found in all protein-coding genes.

• Many different genes and many different types of cells share the same transcription factors

  • Not only those that bind at the basal promoter but even some of those that bind upstream

• What turns on a particular gene in a particular cell is probably the unique combination of promoter sites and the transcription factors that are chosen

Three Stages of Transcription

1. Initiation

   • Recognition step

   • Promoter required

   • Stage completed when DNA strands separate near promoter to form open complex

2. Elongation

   • RNA polymerase synthesizes RNA

   • Template or coding strand used for RNA synthesis

     • Noncoding strand is not used

   • Synthesized 5’ to 3’

3. Termination

   • RNA polymerase reaches termination sequence

   • Causes both the polymerase and newly-made RNA transcript to dissociate from DNA

Inducible Genes

• Inducible genes: genes whose expression is turned on by the presence of some substance

  • Lactose induces expression of the lac genes

  • An antibiotic induces the expression of a resistance gene

• Catabolic pathways

Repressible Genes

• Repressible genes: genes whose expression is turned off by the presence of some substance (co-repressor)

  • Tryptophan represses the trp genes

• Biosynthetic pathways

  • Co-repressor is typically the end product of the pathway

Combinatorial Control

1. Activators: activator proteins stimulate RNA polymerase to initiate transcription

2. Repressors: repressor proteins inhibit RNA polymerase from initiating transcription

3. Modulation: small effector molecules, protein–protein interactions, and covalent modifications can modulate activators and repressors

4. Chromatin: activator proteins promote loosening up of the region in the chromosome where a gene is located, making it easier for RNA polymerase to transcribe the gene

5. DNA Methylation: usually inhibits transcription, either by blocking an activator protein or by recruiting proteins that make DNA more compact