Download DNA transcription > mRNA translation > protein Regions of genes


DNA transcription > mRNA translation > protein

Regions of genes
o Promoter: helps deteremine when the encoded RNA will be made in cell
o Coding region: containes bases read in triplates that dictate AA sequence in
translation

Enzymes of transcription
o RNA polymerase I rRNA (localized to nucleolus)
o RNA polymerase II mRNA (polypeptide coding) and micro RNA (miRNAregulate gene expression) and snRNAs
o RNA polymerase III tRNA and other small RNAs (ex: 5S RNA)
o Contrast: Prokaryotes have ONE RNA polymerase to generate all the types of
RNA (mRNA, rRNA, tRNA) contains 4 subunits α2ββ’ (core enzyme) + σ
factor many σ factors that recognize different promoter regions

Types of RNA
o messenger RNA (mRNA) : carries the message from the DNA to the ribosome
for translation
o microRNA: functions in regulation of gene expression
o ribosomal RNA (rRNA): forms major structural complex of the ribosome with
proteins
o transfer RNA (tRNA): acts as an adapter molecule aligning amino acids with
mRNA sequence held in the ribosome

Directionally of transcription
Template (antiSense)
Coding strand (Sense)

RNA Pol reads 3’ to 5’ and synthesizes in the 5’ to 3’ direction
o Therefore coding strand is what’s read
o Template is what mRNA will look like

Gene recognition
o “Signals” RNA polymerase recognizes are promoters
 promoters contain “boxes” or “elements”= conserved sequence
determine start point and frequency of transcription
 “cis-acting” ---occur in same DNA strand as gene (acting on the same
side)
 “trans-acting”bind and may prevent RNA polymerase from being able to
recognize promoter
o Three classes of promoters
 RNA pol I promoter: two components
 core promoter surrounding the start site and an upstream control
element
 recruited trans-acting transcription factors bind then Pol I binds to
core
 RNA pol II promoter:
 core = short initiator sequence+TATA box about -25 bp of start
site
 a variety of upstream trans-acting factors must also be acquired
 RNA pol III promoter:
 these vary in structure but are loctaed downstream of the start site
(within transcribed region) tRNA transcribed as precursor needing
further processing

mRNA processing
o mRNA made as a precursor protein (hnRNA) then post-transcriptionally
processed to mature form
o Steps:
 removal in introns-splicing of exons
 introns are removed with exons being ligated by spliceosomes
o spliceosome (snRNPs): small nuclear ribonuclear proteins
 addition of 5’cap for stability (added as transcribed)
 Methylated GMP, requires folate and B12
 seals 5’ end and acts as recognition site for ribosome
addition of poly A tail for termination signal
o enzyme=poly A polymerase uses ATP to add As=protein
binding site that can protect mRNA from degradation

rRNA synthesis
o many copies of rRNA genes in nucleolar region
o RNA Pol I will transcribe a 45S transcript, further cleaved into 18S and 28S
rRNA
o 5S rRNA is transcribed outside of nucleoulus by RNA Pol III
o Cytoplasmic ribosome generation
 45S 18S rRNA then complexed with protein=40S small ribosomal
subunit
 45S 28S and 5.8S rRNA + 5S rRNA + proteins=60S ribosomal subunit
 Migrate to cytoplasm via nuclear pores

tRNA synthesis
o tRNA transcribed as a precursor that undergoes post-transcriptional processing
o Steps:
 removal of 5’ prime leader sequence
 replace of two nt at 3’ end with CCA sequence (all tRNA terminate CCA)
 chemical modifications of some bases
 excision of an intron
o





D loop closest to 5’ end = dihydrouridine
Anticodon loop: bases pair with mRNA codon
TC loop: contains ribothymidine and psuedourindine
Variable loop: size varies between 2nd and 3rd loops
CCA end with free Oh at 3’ will bind specific AA
 Attached by nucleotidyl transferase
Comparison of Eukaryotic and Prokaryotic Transcription
1. Eukaryotic (human) cells are diploid: contain 2,000X more DNA that haploid bacteria (3 x 109
bp DNA vs. 4 x 106 bp DNA)
2. Eukaryotic DNA contains introns (non-coding) bacteria do not have introns (hnRNA averages
10X the size of a eukaryotic processed mRNA)
3. Repetitive sequences in eukaryotic DNA
a) DNA has greater complexity that bacterium genome
b) bacterium: single (unique) copy of each gene—most DNA produces
functional product
c) eukaryotic DNA has large amounts of non-coding DNA, multiple
copies of some genes, coding DNA (Approximately 64%), highly
repetitive DNA elements (Alu) moderately repetitive DNA (25%)-rRNA,
tRNA, histone genes
4. Eukaryotic DNA complexes with histones, bacterial DNA no histones
5. Eukaryotic transcription occues in nucleus separated from rest of cell, translation occurs in
cytoplasm. Not true in bacteria transption where translation occurs simultaneously
6. Eukaryotes: one promoter /one gene, bacteria: one promoter/ operon (multiple genes)
7. Prokaryotic transcription start point AT rich (Pribnow or TATA box about -10 nt upstream),
Eukaryotes have Hogness-Goldberg box (TATA box)
8. In bacteria several genes in same metabolic pathway linked into operon, factors can bind and
increase or decrease transcription ( a polycistronic transcript will be produced)
9. Prokaryotes rRNA produce a single large transcript cleaved into 16S, 23S and 5S. tRNA
produced from larger transcript cleavedinto mature forms
o This can cause problem or a solution for Ab tx
o Alpha-amantin blocks eukaryotic transcription
 Mushroom toxin
 Inhibitor of RNA Pol II and mRNA synthesis
 Causes GI imbalance, fever, liver and kidney dysfunction
o Beta-thalassemia
 One of most prevalent (7% carrier frequency) and heterogeneous of
monogenic disorder

Defects in:
 RNA transcription, intron-exon splicing, cap site and RNA
cleavage/processing
To determine hemoglobin status in blood samples supports diagnosis of
anemias and other hemoglobinopathies
1. blood sample
2. lyse RBC
3. expose to oxidizing agent—>converts Fe+2 to Fe+3
4. [Fe+3] calculated by adding CN- or Azide –read on spectrophotometer
Fe+3 color =concentration of Hb in original sample
Infants with thalassemia major are well at birth because of a form of hemoglobin HbF present in
the fetus and newborn. Eventually, this hemoglobin is replaced by mutant Hb. Symptoms emerge
late in the first year of life.
The child develops: irrirability, growth retardation, swelling abdomen (enlarged liver and
spleen) with jaundice and pale skin. Symptoms of severe anemia.
Child becomes dependent on blood transfusions—that create additional problems
like iron overload. Folic acid supplements given.
Treatment: aimed at relieving symptoms if possible/bone marrow transplantation maybe
considered. Future: gene therapy to correct inherited mutation