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 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 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