Chapter 11 Gene Expression: from Transcription to Translation

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10/6/15
Important
how we use our DNA
become organisms and not blobs of cells
Genes and Proteins
Beadle and Tatums hypothesis: was later modified to one gene- one polypeptide chain
one gene can code for multiple proteins through alternative splicing
Flow of Information: Gene Protein
mRNA doesnt live for a long time (minutes to a couple days at most: transient)
this is good to change what is being made
transcription
DNA to RNA
Translation
RNA to protein
Classes of RNA
mRNA:
rRNA: fold on itself, catalyzes chemical RXNs in which amino acids linked to one another
tRNA: translate info in the mRNA code into amino acids, transfers the rRNA
Overview
Transcription
DNA dependent RNA polymerase, responsible for transcription
need the DNA template strand
promoter: sequence just before the gene, RNA polymerases bind to DNA and start transcription
use the same set of information differently in our tissues
RNA grows in the 5 to 3 end, has to go from one letter to the next and stay attached to the DNA
template strand
2 phosphate groups, bond breaks to get 2 inorganic phosphates
lots of RNA polymerase working on a gene at the same time
there is some spacing, because you unwind the DNA then open it up, have space so they can
all unwind properly
unwind in local region, then the area behind it will spontaneously reseal itself
RNA polymerase
add new nucleotides, and correct them when it messes up
Techniques to follow RNA polymerase
took RNA polymerase and attached it to a cover slip, then took DNA molecule and attached a
styrofoam bead, and tracked the bead in place
laser from top and bottom, and the sides, front and back
figure out how much force the RNA polymerase was exerting on the bead in a pulling force and
goes 1 base at a time
Transcription in Bacteria
1 type of RNA polymerase
5 subunits, has a core (holo) enzyme, quaternary structure and pentamer
cells have to have a sigma factor, small protein subunit that helps the polymerase attach to the
DNA
look for signal sequences before the gene begins
10 bases before your gene will have a TATA box
Consensus Sequence
most commom set of letters in that sequence
Pribnow box may help regulate gene expression

e. Terminator sequences typically fold into a hairpin loop that causes the RNA polymerase to
release
i.
might need a rho factor protein or might not?
ii.
so much variation
8. 3 Types of Eukaryotic Polymerases
a. rRNAs are transcribed by RNA polymerase I
b. mRNAs transcribed by RNA polymerase II
c. tRNAs are transcribed by RNA polymerase III
9. RNA polymerases in Eukaryotes
a. transcription factors regulate the activity of RNA polymerases
b. newly transcribed coming out of eukaryotes are processed
i.
almost immediately translated after transcription in bacteria
c. primary transcript (pRNA) initial RNA that gets transcribed, initial raw everything like exons,
introns and everything else
d. transcription unit is the DNA segment corresponding to primary transcript
e. comething
i.
does not go into the cytoplasm at all, only noticed after looking at splicing
10. rRNA
a. rough ER is a myth, ribosomes are not permanently attached to the ER, start off as free
ribosomes, mRNA required to get it to the ER
b. rDNA is the DNA sequence encoding rRNA
c. in nondividing cells, nucleoli, rRNA is being made where ribosomes are produced
i.
cells make a lot of ribosomes, get a lot in the nucleus
ii.
found on multiple chromosomes
d. Nucleoli
i.
3 regions:
1. GC: dark grey area, rRNA that have been made assembling into ribosomes
2. FC: white spot in middle, DNA that codes for the rRNA
3. DFC: dark border around edge of white spot, transcription is occurring
11. rRNA Gene Arrangement
a. tandem repeats: 2 copies back to back
b. nontranscribed spacer sequence,
c. ribosome genes, inside have differenet sequences, parts get edited out later,
i.
3 exons that are internal spacers,
d. 45S, hard to use molecular weight, same is true of nucleotides, S refers to where it falls ina
sucrose gradient
12. rRNA Precursor Synthesis
a. lots of nucleoli making a lot or frobosomes
b. zoom in, see interesting structures, and look like christmas trees, the farther you go down the
gene, the longer the messenger RNA is, so it gets longers are you go further down the DNA
13. Processing the rRNA Precursor
a. takes less than an hour
b. first part is very fast: starting with a 45 S pre-rNA
i.
chop off ETA sequences
ii.
trims sequence to 42-S lighter
1. cant see the 42 S peak, its so close it wouldnt show up
iii.
clip beginning or end of first ITS, separates 18S rubosomal subunit, leftover is 32S
iv.
rRNA start to diffuse out of the nucleous and go into the cytoplasm (10-40 mins)
v.
cleave at the end of the 2nd ITS; separates 28S and 5S left
vi.
trim off ITS pieces from subunits

14. rRNA synthesis and Processing

a. transciption of rRNA precursor
b.
c. cleave at sites 1 and 5
d. cleave at either beginning or end of ITS
e. cleave at site 4
f. last little bit it to trim off ITS sequences
15. tRNA Gene Arrangement
a. tRNAs
i.
located in small clusters scattered around the genome
ii.
have promoter sequences within ght eocding region of the gene (transcribed by poly. III)
iii.
during processing, the tRNA
16. Structure of tRNAS
a. amino acid attached to 3 end of molecule, has a T arm and a D arm
b. secondary structure: assumes clover leaf shape, thumb might be present??
c. 3 nucleotides that match up with the ones on the mRNA
d. Y = any pyrimidine, M3C = 3-methylcytosine or 3-methylguanine
e. folds on itself again on a diagonal access to get an L shape
f. on the mRNA, it would be antiparallel to the genes
17. Wobble in the interaction between codon and anticodon
a. wobble synthesis: suggests than a tRNA can recognize mRNA codons with variable third bases
b. 3rd potiion: not as important
c. base 2: most important for which amino acid is which!
d. 5 GAA matches with complementary 3 UGG
18. Amino Activation
a. AUG is the start codon
b. aminoacyl-tRNA synthesases (aaRS)
i.
add amino acids onto the tRNAs
ii.
energy from ATP is used to give amino acid higher energy state then transferred onto the tRNA
iii.
codons of the mRNA are interpreted according to the recognition abilities of the aaRS
19. Synthesis and Processing of mRNAs
a. precursor of mRNAs are represented by diverse RNAs called heterogenous nuclear RNAs
(hnRNAs)
b. hnRNAs are found only in the nucleus, have large molecular weights, degraded after a short
time
c. lasted effort to make something big then get rid of it
20. PRomoters: Binding Sites for RNA polymerases
a. RNA polymerase II is assisted by general transcription factors (GTFs) to form the preinitiation
complex (PIC)
b. critical portion of the promoter lies 24-32 bases upstream from the initiation site and contains
the TATA box
c. no universal core promoter elements
d. preinitiation complex of GTFs and polymerase assemble at the TATA box
e. downstream promoter elements to promote gene expression
10/8/15
21. Assembly of the Preinitiation Complex
a. TATA binding protein needs to bind at the promoter element (initiator, TATA box, etc)
i.
before this nothing happens at all
b. conformation change to DNA

c.
d.
e.
f.

binding of TFIID sets stage for assembly of the complete PIC

3 GTFs bound to the promoter allows the binding of RNA to polymerase with TFIIF
TFIIE & TFIIH
as long as TFIIH remains bound to the promoter, additional RNA polymerases may be able to
attach for additional rounds of transcription
22. Preinitiation Complex Machinery
a. 80 degree bend in the DNA, and make a loop
b. transcription factors help TATA binding protein
23. Transripction
a. always will give us the coding strand
b. 5 and 3 matter
i.
DNA read 3 to 5 for the mRNA
c. RNA uses uracil and not thymine
24. Structure of mRNA
a. mRNAs share vertain properties
i.
each code for specific polypeptide
ii.
found in cytoplasm
iii.
attatched to ribosomes when translated
iv.
most have noncoding segment
v.
eukayotic mRNAs have modifications at their 5 guanine cap and a 3 poly(A) tail
1. longer poly(A) tail the longer it can live in the cytoplasm
25. Discovery of Split Genes
a. created nuclear RNA
b. DNA regions between these blocks (intervening sequences) are missing in corresponding
mRNA
c. introns: take info out, extron: info that is coding and important
26. Processing EUkaryotic MEssenger RNAs
a. RNA transcripts become associatedwith ribonucleoproteins as they were synthesized
b. during processing 5cap and poly(A) tails are added
c. intervening sequences .
27. Processing Eukaryotic mRNA
a. 5 end of pre-mRNA binds to enzyme with 2 active sites
b. triphosphatase removes terminal phosphate
c. guanyltransferase adds backwards guanine with 5-->5 link
d. methylransferases add CH3 to the guanin, creating 5 methylguanosine cap
e. end of transcription, one enzyme trims off 3 end and adds new on and next enzyme adds a new
poly(A) tail
i.
up to 250 Adenines
28. Synthesis and Processing of mRNAs
a. RNA transcripts become associated wihth ribonuleoproteins as synthesized
b. 5 cap and poly(A) tail added
c.
29. RNA SPlicing
a. breaks and introduced at the 5 and 3 ends (splice sites)
b. sequences between exon-intron boundaries are highly conserved
c. sequence most commonly found at the boundary is g/GU at the 5 end and AG/G at the 3 end
30. RN splicing: Intron Removal
a. breaks are introduced at the 5
b. f
c. f

31. RIbozymes: Self-Splicing RNA

a. mechanism od RNA splicing has led to the study of ribozymes
b. thought to have evolved from self-splicing RNAs
i.
creates a loop, exon 1 and 2 bind together and get rid of the intron
c. ex: group 2 intron
d. mitochondria and chloroplasts have group 1 introns/ self splicing
32. Splicing Pre-mRNA
a. isnt capable of selfsplicing
b. requires the help of small nuclear RNAs
c. processing occurs as each intron becomes associted with a complex called spliceosome
d. spliceosome consists of
33. RNA Splicing: intron Removal
a. NEED
i.
nuclear RNA
ii.
several snRNPs
1. Each snRNP contains a dozen or more proteins, such as the sM protein family
b. snRNAs may be catalytically active components of the snRNPs, not the proteins based on:
i.
pre-mRNA catalyzed by the same pair of chemical rxns
ii.
snRNAs required for splicing pre-mRNA resemble group II introns
c. proposal that combined action of both RNA and a protein in the spliceosome catalyze the 2
chemical rxns required for RNA splicing
34. Splicing Process
a. U1 comes in and binds to 5 end of intron
b. U2 comes in and binds to the U part of the middle of the itnron
c. U4, U5, and U6 come in and binds to exons at 5 end, binds to U2, and a shuttle effect and
things shift
i.
U4 gets dissociated
ii.
U1 leaves
d. first cleavage occurs, glues 5 of intron to some sequence
e. U5 reassociated with U2 and U6 and exon 1 and 2 will bind to each other
35. Why Splicing is important
a. not as important in bacteria
b. from an RNA based world or life form
c. viruses might have originated from living organisms
d. RNA earliest molecule to store info and edit information on its own
36. d
37. small rNAs that regulate gene expression
a. mRNAs
i.
play regulatory role in development
ii.
patterning of the nervous system
iii.
control of cell proliferation and cell death
iv.
leaf and flower development in plants
v.
differentiatoin in various cell types
vi.
there are roles for miRNAs in the development of cancer
b. Some implicated in immune system function
38. Translation
a. most complex activity of the cell
b. 3 main types of RNA
c. use 20 different amino acids
d. similar between prokaryotes and eukaryotes

e. 3 parts
initiation
1. very long process
ii.
elongation
iii.
termination
39. Initiation
a. begins with initiation codon AUG
b. Prokaryotes
i.
Shine-Delgarno sequence repeat of AGGAGG
ii.
scan mRNA in search of start codon
iii.
requires initiation factors (IF)
1. IF1: promotes binding of robosome to mRNA sequence and scan to look for codon
2. IF3: keeps large ribosomal unit together before it tries to assemble
c. Eukaryotes
i.
ACCAUGG sequence
ii.
initiation factors
1. EIF3: same as in bacteria (IF3)
2. EIF1: transfer RNA assemble with large ribosomal subunit
3. EIF2: helps drag all of it in and associate large robosomal subunit and mRNA when it is ready
4. eIF4F: unzip double stranded reagion and straighten it
5. eIF%: helps large ribosomal subunit stay associated
40. Initiation in Bacteria
a. 1) small ribosomal subunit finds mRNA and hunts for the start codon
b. 2) transfer RNA carries first amino aid (methionine)
i.
this MET is modified, has 2 mods, extra formal group and extra methyl
c. 3) tRNA bound and large ribosomal subunit assembles on top of it GTP broken into
GDP
41. Initiation in Eukaryotes
a. requires at least 12 IFs with a total of 25 polypeptide chains
b. 1) small ribosomal and tRNA assembles before hooks up to the mRNA
c. 2) form preinitiation process, first transfer RNA is docked
i.
eIFS 1-3
d. 3) mRNA binds and eIF4F and other initiation make loop structure to relieve supercoiling
e. 4) 43S scans for the AUG start codon
42. Ribosome
a. A site: test codon and anticodon to make sure match
b. P site: peptide bond forms between amino acids
c. E site: exit site
d. gets each tRNA and makes sure they match
e. codon and anticodon match, tRNA mRNA and amino acid shift to the P site, and the peptide
bond is catalyzed
f. goes to the exit site
43. Elongation
a. peptide bond forms
b. tRNA in P site, tRNA2 comes into the A site, brings the amino acids close enough together so
that they interact with each other, bond forms between N and C that are close to each other
c. can make about 10 amino acids per second, fairly slower than transcription
d. ribosome moves along in the 5 to 3 end
e. simpler process, driven by slight conformational change, produces 6 degree rotation in the
ribosomal subunit and moves 3 nucleotides at a time
i.

44. Termination
a. occurs when hit 1 of 3 stop codons
b. not corresponding RNAS, keeps searching for an amino acid to match, need ribosome to go
away and start another protein
c. eukaryotic cells: release factors 1 and 3 work together to recogniz stop codon and help
ribosome to disassemble
d. prokaryotes: get polypeptide on the tRNA to break apart then break ribosome free of mRNA
45. mRNA Surveillance and Quality Control
a. nonsense mutation: produce stop codons and cause premature chain termination
b. nonsense-mediated decay (NMD): nonsense mutations destroyed
c. NMD protects from nonfunctional proteins
d. eon-junctions complex (EJC) used to detect unappropriate stop
46. Visualizing transcription and translation
a. bateria: transcription and translation are coupled and occured simultaneously
b. protein synthesis in basteria startes before mRNA is finished