Transcript Chapter 26 - RNA Metabolism

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
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
DNA-dependent RNA Polymerase
• RNA polymerase (RNA pol) catalyzes DNAdirected 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 50-90 nucleotides/sec (~ 1/10th rate of DNA
replication)
• Growing RNA molecule “peels off” of DNA as it
elongates
• DNA reforms double strand
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'
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
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
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
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
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)
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