1. Chapter 26 - RNA Metabolism
Focus of this chapter is transcription
Three main types of RNA
(1) Transfer RNA (tRNA)
Carries amino acids to translation machinery
Very stable molecules
(2) Ribosomal RNA (rRNA)
Makes up much of the ribosome
Very stable, majority of cellular RNA
(3) Messenger RNA (mRNA)
Encodes message from DNA to ribosomes
Rapidly degraded by nucleases
There are many other RNAs:
- genome of some viruses
- specialized catalysts
- regulatory functions

2. RNA synthesis
Similarities to DNA synthesis
-directionality (5'→3‘)
-steps- initiation, elongation, termination
-copied from a template
Differences from DNA synthesis
No primer required
Only discrete segments of DNA template copied
Only one strand of DNA serves as template
Different set of enzymes
Uracil, ribose

3. DNA-dependent RNA Polymerase
RNA polymerase (RNA pol) catalyzes DNA-directed RNA synthesis (transcription)
RNA pol is core of a larger transcription complex
Complex assembles at one end of a gene when transcription is initiated
First nucleotide triphosphate keeps all 3 phosphates
DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain (about 17 bp at a time) transcription bubble
Mechanism of elongation reaction almost identical to that for DNA polymerase
Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template
New phosphodiester bond formed, PPi released
Adds nucleotides/sec (~ 1/10th rate of DNA replication)
Growing RNA molecule “peels off” of DNA as it elongates
DNA reforms double strand

4. Transcription Initiation
Transcription complex assembles at an initiation site (DNA promoter region)
Short stretch of RNA is synthesized
Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes
Eukaryotic genes each have their own promoter
Transcription of E. coli ribosomal RNA genes
Genes have a 5'→3' Orientation
Convention for double-stranded DNA: Coding strand (non-template)(top) is written: 5'→3' Template strand (bottom) is written: 3'→5'
Gene is transcribed from 5‘ end to the 3' end
Template strand of DNA is copied from the 3' end to the 5' end
Growth of RNA chain proceeds 5'→3'

5. Transcription Complex Assembles at a Promoter
Orientation of a gene
Transcription Complex Assembles at a Promoter
Consensus sequences are found upstream from transcription start sites
DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promoter site
The s subunit of prokaryotic RNA polymerases is required for promoter recognition and formation of the complex

6. (2) -35 region (-35 bp upstream) from start site
E. coli promoter
(1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp)
(2) -35 region (-35 bp upstream) from start site
A  (sigma) subunit of RNA pol also required
Strong promoters match consensus sequence closely (operons transcribed efficiently)
Weak promoters match consensus sequences poorly (operons transcribed infrequently)
 Subunit increases the affinity of the core polymerase for specific promoter sequences
 Subunit also decreases the affinity of the core polymerase for nonpromoter regions
Core polymerase (no  subunit) binds DNA nonspecifically

8. RNA Polymerase Changes Conformation
Unwinding of DNA at the initiation site requires a conformational change
RNA pol (R) and promoter (P) shift from:
(RPc) closed complex (DNA double stranded)
(RPo) open complex (18 bp DNA unwound) (forms transcription bubble)
Transcription Termination
Only certain regions of DNA are transcribed
Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences
Two types of termination sequences: (1) Unstable elongation complex (2) Rho-dependent termination

9. Rho-dependent termination sites
Pause sites
Pause sites - regions of the gene where the rate of elongation slows down (10 to 100-fold) or stops temporarily
Transcription termination often occurs here
G-C- rich regions are more difficult to separate than A-T rich regions and may be pause sites
Pause is exaggerated when newly transcribed RNA can form a hairpin
Rho-dependent termination sites
Rho () is a protein factor that triggers disassembly of the transcription complex at some pause sites
Rho binds to ssRNA chain, destabilizing the RNA-DNA hybrid and terminating transcription

10. Transcription in Eukaryotes
Eukaryotic RNA Polymerases
Three different RNA polymerases transcribe nuclear genes
Other RNA polymerases found in mitochondria and chloroplasts
Eukaryotic Transcription Factors
Same reactions as prokaryotic transcription
More complicated assembly of machinery
Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs)

11. RNA polymerase II
Transcribes protein-encoding genes and some small RNA encoding genes
Protein-encoding RNA synthesized by RNA pol II is called mRNA precursor (or hnRNA)
Many promotors recognized by RNA pol II contain the TATA box (consensus sequence TATAAA)
General transcription factors interact directly with RNA pol II