Chapter 21
1) Know the central dogma of Biology
2) Know what housekeeping genes are.
3) Know the different type of RNA and there roles in the cell.
4) Know the relationship between RNA steady state abundance, rate of synthesis, and
stability.
5) Know what happens during the different stages of replication
a. Initiation: •Rate limiting step of trxn.
i)
Requires unwinding of DNA and synthesis of primer.
ii)
Conformational change occurs after DNA binding of RNA polymerase
holo-enzyme.
iii)
First RNA Polymerase binds to DNA (closed-complex), then
conformational change in the polymerase (open complex) causes
formation of transcription bubble (strand separation).
iv)
RNA polymerase has two binding sites for NTPs
v)
Initiation site prefers to binds ATP and GTP (most RNAs begin with a
purine at 5'-end)
vi)
Elongation site binds the second incoming NTP
vii)
3'-OH of first attacks alpha-P of second to form a new phosphoester
bond (eliminating PPi)
viii) When 6-10 unit oligonucleotide has been made, sigma subunit
dissociates, completing "initiation“
ix)
NusA protein binds to core complex after disassociation of s-factor to
convert RNA polymerase to elongation form.
b. Elongation:
i)
Polymerase is accurate - only about 1 error in 10,000 bases
ii)
Even this error rate is OK, since many transcripts are made from each gene
iii)
Elongation rate is 20-50 bases per second - slower in G/C-rich regions
(why??) and faster elsewhere Topoisomerases precede and follow polymerase
to relieve supercoiling
c.Termination
i)
Pausing
a. pause sites are GC rich
b. hairpin structures exaggerate pausing
c. Pausing destabilizes the RNA:DNA base pairing in active site leads to
disassembly of transcription complex.
d. Nus A protein promotes pausing at hairpin structure.
ii)
Rho dependent termination
a. rho protein is a helicase
b. unwinds new RNA strand from Dna strand in the transcription bubble.
c. destabilizes the RNA:DNA base pairing in active site leads to disassembly
of transcription complex.
6) E. coli RNA polymerase
a. Know the functions of the beta, beta’ and alpha subunits.
b. Know the function of the sigma factor and how it affects the binding of the RNA
polymerase complex to the promoter.
7) E. coli promoter
a. know what concensus sequences are
b. know the names of the two major concensus sequences associated with the ER.
Coli promoter region (-35 region and the Pribnow box (-10 region)
c. Know that the sigma factor binds to these two regions.
d. Know how the RNA polymerase identifies the promoter (binds to DNA and scans
in both directions)
8) Know that transcriptional initiation is the rate-limiting step in transcription.
1) Eukaryotic Transcription
a. Know the functions of RNA polymerase I, II, and III
b. Know what is the TATA box.
2) Know the difference between a constitutively expressed and induced gene.
3) Know what cis and trans acting elements are.
4) Know the properties of typical cis –elements found in promoters and enhancer
elements.
5) Know the differences between co-avtivators, co-repressors, activators and
repressors.
6) Know the properties of the three major structural classes of DNA binding
proteins.
7) Know that the binding of some DNA binding proteins can be regulated by
allosteric modification
8) Know what is an operon and know its the basic organization.
9) Lac operon
a. Know the organization of the lac operon
b. Know the functions of the three enzymes encoded by the lac operon.
c. Know the general properties of the lac I protein.
d. Understand what is going on the lac I protein binds to an inducer
molecule. Know how that effects transcription of the lac operon.
e. Understand what is happening to the lac operon during catabolite
repression.
10) tRNA processing – know what is happening during tRNA processing. Know
which RNA polymerase in eukaryotes is responsible for transcription of the tRNA
transcript. Know that some bases of the tRNA are chemically modified.
11) rRNA processing – know what is happening during tRNA processing. Know
which RNA polymerase in eukaryotes is responsible for transcription of the rRNA
transcript. Know that the rRNA transcript also encodes tRNAs.
12) mRNA processing in eukaryotes
a. 5’ cap addition – know what type of bases is added to the 5’end of the premRNA during capping . Know that this base is modified by methylation.
Know that the 5’ cap structure is important to the stability of the mRNA.
b. 3’ polyadenylation – Understand the processes involved. Know that once
the RNA polymerase II transcribes the sequence AAUAAA a
endonuclease cleaves the transcribe pre-mRNA 10 to 30 base pairs down
stream from this sequence after which an enzyme know as poly(A)
polymerase adds a string of 100 to 200 A’s to the 3’ end of the mRNA.
Know that the poly(A) tail is important to determining the stability of the
mRNA molecule.
c. Splicing – Removal of introns. Know what are introns and exons. Know
that the 2 nucleotides at the 5’ end of an intron are always GU and the 2
nucleotides at the 3’ end of an intron are always AG. Introns also contain
an internal consensus sequence known as the branch site. Know that a
lariot structure forms by the interaction of the 5’ end of the intron and the
branch site. The lariot structure is then excised and the exons joined.
Know that snRNA are involved in the identification of the intron/exon
junction.