Some nomenclature conventions RNAP RNA DNA Similarities and

Some nomenclature conventions RNAP

RNA DNA

Similarities and Differences Between DNA and RNA • Similar strand structure • Can define a 5’ and 3’ end • 2’ hydroxyl in RNA: causes stability differences) • Uracil in RNA takes the place of Thymine in in DNA

RNA polymerase

Bacterial (Prokaryotic) Transcription • Promoters רצף המכוון את רנא פולימראז תחילת הגן - DNA sequences that guide RNAP to the beginning of a gene (transcription initiation site). • Terminators רצף הגורם לרנא פולימראז להפסיק את תרגום הגן - DNA sequences that specify then termination of RNA synthesis and release of RNAP from the DNA. • RNA Polymerase (RNAP) - Enzyme for synthesis of RNA. • Reaction (ordered series of steps) 1) Initiation. 2) Elongation. 3) Termination.

Other important nomenclature conventions Transcription Initiation Site “Upstream” 5’ 3’ -5 -4 -3 -2 -1 “Downstream” +1 +2 +3 +4 +5 +6 3’ 5’ Direction of transcription Template strand There is no “zero”

RNAP binds a region of DNA from -40 to +20 The sequence of the non-template strand is shown -10 region TTGACA… 16 -19 bp. . . TATAAT “-35” spacer “-10” לאורך ה"ספיסר" יש משמעות – ארוך או קצר יוצר פרומוטר חלש

Sigma factor

Finding and binding the promoter initiation Closed complex formation RNAP bound -40 to +20 Open complex formation RNAP unwinds from 10 to +2 Binding of 1 st NTP Requires high purine [NTP] Addition of next NTPs Dissociation of sigma Requires lower purine [NTPs] After RNA chain is 6 -10 NTPs long

RNA polymerase elongation זיווגי בסיסים 12 - ה"עין" כ

RNA Synthesis is in the 5’ to 3’ Direction DNA strand RNA strand Subsequent hydrolysis of PPi drives the reaction forward OH OH RNA has polarity (5’ phosphate, 3’ hydroxyl)

Rho-Dependent Transcription Termination (depends on a protein AND a DNA sequence) G/C -rich site RNAP slows down Rho helicase catches up Elongating complex is disrupted

Rho-Independent Transcription Termination (depends on DNA sequence - NOT a protein factor) Stem-loop structure

Rho-independent transcription termination • RNAP pauses when it reaches a termination site. • The pause may give the hairpin structure time to fold • The fold disrupts important interactions between the RNAP and its RNA product • The U-rich RNA can dissociate from the template • The complex is now disrupted and elongation is terminated

RNA polymerase

Ways to Regulate Transcription Alternate sigma factor usage: controls selective transcription of entire sets of genes vegetative (principal s) s 70 (16 -19 bp) TTGACA heat shock nitrogen starvation s 32 s 60 +1 TATAAT (5 -9 bp) A +1 CNCTTGA (13 -15 bp) CCCATNT (5 -9 bp) A CTGGNA (6 bp) TTGCA +1 (5 -9 bp) A

Pre-m. RNA

5’UTR 3’UTR

שלבי הטרנסקריפציה Initiation • Elongation • Termination •

YSPTSPS The number of these repeats varies; the mammalian protein contains 52, while the yeast protein contains 26. Serine 5 phosphorylation is confined to promoter regions and is necessary for the initiation of transcription, whereas Serine 2 phosphorylation is important for m. RNA elongation and 3'-end processing

Stages in Initiation of Transcription • Bacterial transcription • Closed complex: holoenzyme+promoter • Open complex (DNA melting, not need ATP) • Abortive transcripton • Productive initiation – Transcribe past +9 – Sigma dissociates • Elongation • Eukaryotic transcription • Preinitiation complex (PIC) assembly • PIC activation (DNA melting, needs ATP) • Abortive transcription • Productive initiation – CTD phosphorylated – Promoter clearance • Elongation

Elongation NTPs

TERMINATION • RNA polymerase meets the terminator • Terminator sequence: AAUAAA • RNA polymerase releases from DNA • Prokaryotes-releases at termination signal • Eukaryotes-releases 10 -35 base pairs after termination signal

transcription

m. RNA מהלך חיי מולקולאת Transcription regulation

a steroid hormone

Gene expression

Basics of cell biology: development Fertilized egg

Basics of cell biology: development Cells differentiation is due to different gene Switches going on and off These cells are different because they express a subset of their 24, 000 genes

m. RNA localization Spector

Promoter Gene To specify a new cell: place it in the right environment or

EGF Hedgehog Wnt Promoter Gene Retinoic acid FGF HGF To specify a new cell: place it in the right environment or Substitute drugs/proteins for environment Mophogens