RNA Transcription and Processing

1
RNA Transcription and Processing
Hartl 1999
Sections 9.2 - 9.4
2
Review

• Gene expressionphenotype determined by
  production of certain molecules
• These molecules are known as polypeptides,
  commonly referred to as proteins.
• Proteins are made of amino acids as specified by
  DNA sequence
• DNA?RNA?Gene Product (polypeptide/protein)
• Gene expression Genotype ? Phenotype

3
RNA

• RNA-ribonucleic acid
• Contains the sugar ribose
• Ribonucleoside triphosphates (ATP, GTP, CTP,
  UTP) Uuracil

• Single DNA strand serves as template
• RNA polymerase for synthesis

Hartl 1994
4
Colinearity

• Nucleotide sequence of DNA determines the linear
  order of amino acids in a polypeptide (protein)
• Discovered by Charles Yanofsky in 1950s
• Examined the trpA (tryptophan synthase) gene in
  E. coli

5
Colinearity

• Point-to-point correspondence
• Universal in prokaryotes
• Intervening non-coding sequences in most
  eukaryotic genes, known as introns
• Introns generally do not affect gene function or
  the order of amino acids in proteins.

6
Template DNA Sequence
Prokaryotic DNA
TATAATATACCAGCCTGCCGTCCGTTCT
NON-CODING REGION (INTRON)
TERMINATION SEQUENCE
PROMOTER
CODING REGIONS (EXONS)
TATAATATACCAGCCTGCCCCGTATGTCCGTTCT
Eukaryotic DNA (single strand)
7
Transcription DNA?RNA
Prokaryotic DNA
1
TATAATATACCAGCCTGCCGTCCGTTCT
3
5
RNA transcript
UAUGGUCGGACGGCAGGCAAGA
3
5
Each group of 3 nucleotides is called a
codon. Promoter is not transcribed.
8
Transcription DNA?RNA
Eukaryotic DNA
5
3
1
TATAATATACCAGCCTGCCCCGTATGTCCGTTCT
RNA transcript
UAUGGUCGGACGGGGCAUACAGGCAAGA
5
3
Each translated codon will specify an amino acid.
Note that the intron is transcribed.
9
Promoter Recognition

• Promotera specific DNA sequence where RNA
  polymerase binds
• Most promoter sequences have common
  motifs?consensus sequence
• An example, TATA box TATAAT

TATAATTATGTTCATGATTTAACTTAGGTTTAACTCTATGGTTAGACTTA
TAAT
modified from Hartl 1999
10
Expression/Chain Initiation

• Extent of expression depends on
• binding strength of promoter region (prokaryotes)
  
• presence of enhancers (eukaryotes)
• Transcription actually begins at a nearby site,
  denoted as 1

11
Chain Elongation

• RNA chains elongate in the 5? 3direction
• RNA is complementary and antiparallel to the DNA
  template strand

Hartl 1999
12
Phosphodiester Bonds
Modified from Hartl 1994

F
F
RNA polymerase forms the phosphate-sugar bonds
between adjacent nucleotides
F
F
F
U
13
Chain Elongation

• RNA polymerase unwinds short regions of the the
  DNA duplex
• less than 20 base pairs unwound at any time

• DNA duplex reforms after RNA polymerase passes

Hartl 1994
14
Chain Termination

• Transcription-termination sequences in the DNA
  terminate RNA synthesis
• RNA polymerase dissociates from DNA
• Newly formed RNA strand is released

15
Chain Termination

• Example inverted repeats (see Figure 9.9)
• Self-termination most common
• Presence of a termination protein sometimes
  required

16
Multiple Transcription

• DNA may be transcribed multiple times
• Multiple transcription events may occur
  simultaneously along the same DNA template strand

Hartl 1999
17
Fate of Transcripts

• In prokaryotes, the primary transcript is
  messenger RNA (mRNA)
• Few modifications necessary

• In eukaryotes, the RNA molecule must be processed
  before becoming mRNA
• Introns must be removed

18
mRNA

• Usually contains a leader sequence at the 5 end,
  which is not translated
• Coding sequence is translated portion
• Specifies the amino acid sequence of the
  polypeptide/protein chain
• Typically between 500 and 3000 bases long, but
  may be longer

19
mRNA

• Short lifetime in prokaryotes
• degraded within minutes of synthesis
• Longer lifetime in eukaryotes
• minutes to several days
• Molecules not needed are degraded
• Nucleotides are recycled

20
RNA Processing

• 5 end methylguanosine cap added

GUAUGGUCGGACGGGGCAUACAGGCAAGAAAAA

• 3 end poly-A tail added

21
RNA Processing

• RNA splicing occurs in spliceosomes to remove
  non-coding sequences (eukaryotes)

GUAUGGUCGGACGGGGCAUACAGGCAAGAAAAA
Splice sites
GUAUGGUCGGACGG CAGGCAAGAAAAA
Intron
GGCAUA
22
RNA Processing

• RNA splicing occurs in spliceosomes to remove
  non-coding sequences (eukaryotes)

GUAUGGUCGGACGGGGCAUACAGGCAAGAAAAA
Splice sites
GUAUGGUCGGACGGCAGGCAAGAAAAA
mRNA
Intron
GGCAUA
23
RNA Processing

• Spliceosomes--found in nucleus
• protein and small nuclear ribonucleoprotein
  particles (snRNPs)
• specificity

• RNA splicing--consult figure 9.14 (pg. 316) for
  more detailed description of donor splice site,
  branch site, acceptor splice site, and lariat
  formation

24
Summary

• RNA synthesis
• RNA polymerase required
• ATP, CTP, GTP, UTP precursors
• Promoter region (usually consensus sequence) for
  polymerase binding
• Chain elongation in the 5? 3direction with
  phosphodiester bond formation
• Transcription-termination sequences halt RNA
  synthesis alone or with special proteins

25
Summary

• Primary transcript is messenger RNA (mRNA) in
  prokaryotes must be modified to become mRNA in
  eukaryotes
• 5 end methylguanosine cap, 3 end poly-A tail
• Splicing occurs to remove non-coding sequences
  known as introns in eukaryotes
• occurs in spliceosomes

26
Summary

• DNA nucleotide sequence determines RNA sequence
• In prokaryotes point-to-point correspondence is
  universal
• In eukaryotes the sequence is interrupted by
  introns which are later removed
• RNA transcripts become mRNA which are translated
  in polypeptide synthesis