Gene Expression: Transcription

1
Gene ExpressionTranscription

• Paul D. Brown, PhD
• paul.brown_at_uwimona.edu.jm
• BC21C Molecular Biology I

2
GENE EXPRESSION

• Transcription and translation are the two main
  processes linking gene to protein
• Genes (on DNA) provide the instructions for
  making specific proteins.
• The bridge between DNA and protein synthesis is
  RNA (messenger RNA)

3
Learning Objectives

• Describe the process of transcription
• Describe the function of the following
• mRNA 3' and 5' ends
• RNA polymerase Promoter
• Coding sequence Transcription
• Codon terminator
• Template strand
• Define the following
• Introns Exons
• Precursor mRNA Cap
• Poly-A tail Mature mRNA
• Describe the significance of splicing and
  post-transcriptional processing

4
Gene Expression in Bacteria
5
Gene Expression in Eukaryotes
6
(No Transcript)
7

• Within the first half of 1960s the entire code
  was deciphered.

8
The Genetic Code

• The genetic code is redundant but not ambiguous.
• Codons synonymous for the same amino acid often
  differ only in the third codon position (wobble
  theory).
• To extract the message from the genetic code
  requires specifying the correct starting point.
• In summary, genetic information is encoded as a
  sequence of non-overlapping base triplets, or
  codons, each of which is translated into a
  specific amino acid during protein synthesis.

9

• The genetic code is nearly universal, shared by
  organisms from the simplest bacteria to the most
  complex plants and animals.
• In laboratory experiments, genes can be
  transcribed and translated after they are
  transplanted from one species to another.
• Transgenic tobacco plant expressinga firefly
  gene.

10
Information transfer- Prokaryotes
11
Information transfer- Eukaryotes
12
Transcription is the DNA-directed synthesis of
RNA a closer look
13
Bacterial RNA Polymerase

• Bacteria have only one RNA polymerase
• Active enzyme is a pentamer containing 4
  different polypeptide chains (total MW 500 kDa)
• b (155 kDa)
• b (151 kDa)
• s70 (70 kDa)
• a (36.5 kDa)
• Holoenzyme a2bbs70 ? a2bb s70

14
Comparison of E. coli RNA pol with DNA PolI and
PolIII
RNA pol DNA pol I and III
Similarities DNA-template-directed Requires 4 NTPs Requires divalent cations Yes Yes (rNTPs) Yes Yes Yes (dNTPs) Yes
15
Comparison of E. coli RNA pol with DNA PolI and
PolIII
RNA pol DNA pol I and III
Differences Function Initiates chains Terminates chains Recognizes sequences Uses intact duplex template Product Proofreading Transcription Yes Yes Yes Yes ss RNAs ? Replication/repair No No No No ds DNA strands Yes
16

• Transcriptioncan beseparatedinto
  threestagesinitiation, elongation,
  andtermination

17

• As RNA polymerase moves along the DNA, it
  untwists the double helix, 10 to 20 bases at time.

18

• Transcription proceeds until after the RNA
  polymerase transcribes a terminator sequence in
  the DNA.
• In prokaryotes, RNA polymerase stops
  transcription right at the end of the terminator.
• Both the RNA and DNA is then released.
• In eukaryotes, the polymerase continues for
  hundreds of nucleotides past the terminator
  sequence, AAUAAA.
• At a point about 10 to 35 nucleotides past this
  sequence, the pre-mRNA is cut from the enzyme.

19
Eukaryote RNA polymerases

• Three nuclear RNA polymerases
• polI found in nucleolus synthesizes pre-rRNA
• polII found in nucleoplasm synthesizes hnRNA
  and mRNA
• polIII found in nucleoplasm synthesizes
  pre-tRNA and 5S RNA
• Mitochondrial synthesizes mtRNA
• Chloroplast synthesizes ctRNA

20

• In eukaryotes, proteins called transcription
  factors recognize the promoter region, especially
  a TATA box, and bind to the promoter.

21
Eukaryotic cells modify RNA after transcription

• At the 5 end, a modified form of guanine is
  added, the 5 cap.
• At the 3 end, an enzyme adds 50 to 250 adenine
  nucleotides, the poly(A) tail.
• The mRNA molecule also includes nontranslated
  leader and trailer segments.

22

• RNA splicing removes introns and joins exons to
  create an mRNA molecule with a continuous coding
  sequence.

23

• RNA splicing appears to have several functions.
• First, at least some introns contain sequences
  that control gene activity in some way.
• Splicing itself may regulate the passage of mRNA
  from the nucleus to the cytoplasm.
• One clear benefit of split genes is to enable a
  one gene to encode for more than one polypeptide.
• Alternative RNA splicing gives rise to two or
  more different polypeptides, depending on which
  segments are treated as exons.
• Early results of the Human Genome Project
  indicate that this phenomenon may be common in
  humans.

24

• Split genes may also facilitate the evolution of
  new proteins.
• Proteins often have a
• modular architecturewith discrete
  structuraland functional regionscalled domains.
• In many cases, different exons code for
  different domains of a protein.

25
Mixing and matching produces antibody diversity
26
Inhibition of Splicing

• Systemic Lupus Erythematosus
• Chronic, multi-system autoimmume disease
• Affects mostly women, 20-60 years
• Clinical features
• Skin rashes, arthritis, kidney damage
• Auto-antibodies attack nucleus of the cell

27
Post-transcriptional processing

• Thalassemias due to defect in mRNA synthesis
• a-thalassemia deficiency in a globin chains
• ß-thalassemia deficiency in ß globin chains
• Results in anemia at ca. 6 mths- x HbF ?HbA

28
rRNA Processing
Prokaryotes
Eukaryotes
29
Eukaryotic tRNA processing

• Often transcribed in the intragenic (spacer)
  regions or at the distal end of rRNA genes
• Processing involves
• Folding
• Endonucleolytic cleavage (RNaseF at 3 end and
  RNaseP at 5 end)
• Exonuclease processing (RNaseD) exposes the CCA
  3-end
• Base modification

30
Inhibitors of Transcription

• Bind to DNA
• Actinomycin D (from Streptomyces)
• Intercalates between 2 GC base pairs
• Does not greatly impair binding of RNA
  polymerase however, blocks chain elongation (cf.
  ethidium bromide)
• Bind to RNA polymerase
• Rifampicin (synthetic derivative of rifamycin)
• Binds to bacterial RNA polymerase (? subunit)
• Used in treatment of TB
• a-Amanitin (from death cap or destroying
  angel mushroom)
• Binds to RNA pol II to less extent, pol III
  (eukaryotes)
• Incorporated into growing RNA chain
• Cordycepin (substrate analog)
• Causes chain termination after incorporation no
  OH group