Transcript Chapter 19 Nucleic Acids

Protein Synthesis
• Genome - the genetic information of an organism
• DNA – in most organisms carries the genes
• RNA – in some things, for example retroviruses
like the AIDS virus
• Gene - a DNA sequence that is transcribed
(includes genes that do not encode proteins)
Information specifying protein
structure
• Transcription - copying of the DNA sequence
information into RNA
• Translation - Information in RNA molecules is
translated during polypeptide chain synthesis
• Information flow: CENTRAL DOGMA
• DNA
RNA
PROTEIN
Biological information flow
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
Biological information flow
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
• DNA is continuously unwound as RNA pol
catalyzes a processive elongation of RNA chain
The Chain Elongation Reaction
• Mechanism almost identical to that for DNA
polymerase
• Growing RNA chain is base-paired to DNA
template strand
• Incoming ribonucleotide triphosphates (RTPs)
form correct H bonds to template
• New phosphodiester bond formed, PPi released
RNA polymerase reaction
• Catalyzes polymerization in 5’
3’ direction
• Is highly processive, and thermodynamically
assisted by PPi hydrolysis
• Incoming RTPs: UTP, GTP, ATP, CTP
• Growing single-stranded RNA released
• Adds 30-85 nucleotides/sec (~ 1/10th rate of
DNA replication)
• RNA
polymerase
reaction
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
A. Genes have a 5’
Orientation
3’
• Convention for double-stranded DNA:
Coding strand (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
Promoter sequences from 10
bacteriophage and bacterial genes
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
• Strong promoters match consensus sequence
closely (operons transcribed efficiently)
• Weak promoters match consensus sequences
poorly (operons transcribed infrequently)
Initiation of transcription
in E. coli
(two slides)
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
Transcription in Eukaryotes
A. Eukaryotic RNA Polymerases
• Three different RNA polymerases transcribe
nuclear genes
• Other RNA polymerases found in
mitochondria and chloroplasts
• Table 21.4 (next slide) summarizes these
RNA polymerases
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)
Transcription of Genes Is
Regulated
• Expression of housekeeping genes is constitutive
• These genes usually have strong promoters and
are efficiently and continuously transcribed
• Housekeeping genes whose products are required
at low levels have weak promoters and are
infrequently transcribed
• Regulated genes are expressed at different levels
under different conditions
Role of regulatory proteins in
transcription initiation
• Regulatory proteins bind to specific DNA
sequences and control initiation of transcription
• Repressors - regulatory proteins that prevent
transcription of a negatively regulated gene
• Negatively regulated genes can only be
transcribed in the absence of the repressor
• Activators - regulatory proteins that activate
transcription of a positively regulated gene
Inducers and corepressors
• Repressors and activators are often allosteric
proteins modified by ligand binding
• Inducers - ligands that bind to and inactivate
repressors
• Corepressors - ligands that bind to and
activate repressors
• Four general strategies for regulating
transcription
• Strategies for
regulating
transcription
initiation by
regulatory
proteins
Posttranscriptional Modification of
RNA
• Mature rRNA molecules are generated in both
prokaryotes and eukaryotes by processing the
primary transcripts
• In prokaryotes, 1o transcripts often contain
several tRNA precursors
• Ribonucleases (RNases) cleave the large
primary transcripts to their mature lengths
Ribosomal RNA Processing
• Ribosomal RNA in all organisms are produced
as large primary transcripts that require
processing
• Processing includes methylation and cleavage
by endonucleases
• Prokaryotic rRNA primary transcripts ~30S
• Contain one copy each: 16S, 23S, 5S rRNA
Eukaryotic mRNA Processing
• In prokaryotes the primary mRNA transcript is
translated directly
• In eukaryotes transcription occurs in the
nucleus, translation in the cytoplasm
• Eukaryotic mRNA is processed in the nucleus
without interfering with translation
• In some mRNA, pieces are removed from the
middle and the ends joined (splicing)
Eukaryotic mRNA Molecules
Have Modified Ends
• All eukaryotic mRNA precursors undergo
modifications to increase their stability and
make them better substrates for translation
• Ends are modified so they are no longer
susceptible to exonuclease degradation
• The 5’ ends are modified before the mRNA
precursors are completely synthesized
• Guanylate base is
methylated at N-7
• 2- Hydroxyl groups
of last two riboses
may also be
methylated
Poly A tails at the 3’ ends of mRNA
precursors
• Eukaryotic mRNA precursors are also modified
at their 3’ ends
• A poly A polymerase adds up to 250 adenylate
residues to the 3’ end of the mRNA precursor
• This poly A tail is progressively shortened by 3’
exonucleases
• The poly A tail increases the time required for
nucleases to reach the coding region
Some Eukaryotic mRNA
Precursors are Spliced
• Introns - internal sequences that are removed
from the primary RNA transcript
• Exons - sequences that are present in the
primary transcript and the mature mRNA
• Splice sites - junctions of the introns and
exons where mRNA precursor is cut and joined
Triose phosphate isomerase
gene (nine exons and eight
introns)