Molecular Biology (BIOC 3512)

Spring 2014
Professor Manley
Study Guide Exam 1
Note that this does NOT cover all material required for the exam, but rather provides a
framework for organizing many of the mechanistic processes that we have learned. It is
important to treat it as such, and supplement this with your in-class notes from
Professor Manleys lectures, which span the set of topics on which you may be tested.

The RNA world

o Specific Types
mRNA protein coding strands that are spliced (sometimes
alternatively with permuted exonic arrangements) to remove
intervening intronic sequences
tRNA amino acid-binding strand with a characteristic secondary
and tertiary structure, in charge of loading amino acids onto an
elongating polypeptide during translation
rRNA extensively folded strands in complex with proteins, which
collectively form ribosomes
lncRNA - long non-coding RNAs, which can serve a variety of
regulatory or other, uncharacterized functions.
Not required for exam, but one prominent example of this is Xist,
which is an integral effector of the dosage compensation machinery
for females with two sex chromosomes. Xist coats one of the X
chromosomes, arbitrarily chosen, to form a tightly bound Barr body
that is functionally inactive.

uaRNA upstream antisense RNA, which is implicated in

bidirectional transcription. As of yet, the function, if any, remains
unclear, though there are possible detrimental effects relating to
accumulation of these RNA species
miRNA microRNA, which serve an important role in posttranscriptional regulation of gene expression. These miRNAs, when

cleaved, hybridize to mRNAs of complementary sequences to

promote their degradation.
o Two broad categories
Complexity (for humans) number of different kinds of
mRNA/lncRNA > tRNA > rRNA
Mass - rRNA > tRNA > mRNA/lncRNA
Note the inverse relationship between mass and complexity. Why
might this be the case?
o Instability
2 OH on the ribose is capable of attacking the adjacent phosphate,
resulting in autocatalytic cleavage. The 5OH of the adjacent
nucleotide is a good leaving group, knocking it off and creating
separate nucleotides.
o Structure
Many tertiary configurations are possible because it is single
stranded, allowing for considerable flexibility. This allows for both
greater functional potentialities than DNA, which has a relatively
rigid double-stranded helical structure, but also exposes additional
vulnerability due to single strands ability to wrap around and bind to
themselves.
Not required for exam, but for the curious-minded, there is actually
considerable research being done on nucleic acid aptamers, or
oligonucleotide sequences with distinctive tertiary structures that
can bind to specific molecules, for biological and therapeutic
purposes. One interesting example of this in bacteria is a
riboswitch, which is an mRNA segment that can bind small
molecules. The binding event then regulates the efficiency of
translation of the mRNA, in essence providing a means of
autoregulation by the mRNA itself. Pretty neat!

[Prokaryotes] Transcription
o RNA Polymerase (RNAP)
Only one form, unlike three in eukaryotes
Holoenzyme multimeric complex composed of several subunits
that also require a coenzyme, in this case factor, to carry out its
function. factor exchanges the ability of RNAP to non-specifically

bind DNA for promoter specificity to initiate transcription; however,

upon promoter binding, factor is ejected from the complex. This
is because the loss of non-specific DNA binding due to the factor
opposes elongation. What might happen if the factor remained?
o Termination
Intrinsic
Sequence defined RNA structures guide detachment of
the RNA polymerase, without aid from external factors
What actually causes ejection of the RNAP from the DNA
strand though? The immediate thought might be that the
stem loop structure creates a physical barrier that occludes
further passage of the RNAP. However, think of the
sequence of events. For the stem loop structure to have
formed, the RNAP must already be beyond it, preventing it
from physically obstructing passage of RNAP.
Mechanism a protein bound to the RNAP (nusA)
recognizes the stem loop at the same time that the RNAP is
transcribing the polyU tract. These coincident events allow
for precise and easier detachment of RNAP from the DNA.
Extrinsic
Rho helicase travels along behind the RNAP, with a
slight lag. Eventually, Rho ejects the RNAP, which
pauses after it reaches the termination sequence,
completing the termination process itself.

[Eukaryotes] RNA polymerase II (pol II)-dependent transcription

o Responsible for mRNA, snRNA, miRNA, lncRNA, uaRNA
o Promoter four main sequence elements
TATAAA (TATA box)
Inr (initiator element)
BRE (TFIIB response element)
DPE (downstream promoter element)
o Pre-initiation complex
RNA Polymerase II (pol II)
C-terminal domain (CTD) is composed of heptad repeats of
YSPTSPS, prime targets for phosphorylation. Because of
this, the CTD is a hub for localization of many proteins critical

for transcription initiation, as well as a target for regulation of

gene expression.
Transcription factors (TFs)
Associated with promoter recognition
o TFIID composed of TATA binding protein (TBP) and
many different TBP associated factors (TAFs). TAFs
can recognize other core promoter sequences (Inr,
DPE), and can have bromodomains (or even histone
acetyltransferase functions) or chromodomains to
interpet epigenetic instructions for regulating gene
expression
o TFIIB recognizes BRE, stabilizes the TFIID/DNA
interaction by binding the TBP flanking region
o TFIIA stabilizes TFIID
Associated with RNAP/transcription start
o TFIIH dual role in promoter clearance and RNAP
movement. Two functions:
ATP-dependent helicase cleaves - bond
in ATP to power helicase-mediated unwinding
of DNA for promoter clearance
Cyclin-dependent kinase (CDK)
CDK7/Cyclin H phosphorylates CTD at serine 5
to spur transcription
Mediator complex of > 20 polypeptides which provides a scaffold
for contact with other proteins required for activation of
transcription. Stabilizes the pre-initiation complex on DNA, and also
later facilitates removal. The conformational stability provided by
mediator for the pre-initiation complex is critical to activation.
o Regulation of transcription activation
Many genes have the pre-initiation complex loaded but have a
paused RNA pol II
Release of paused pol II
P-TEFb regulates release of the paused polymerase.
Normally it is bound to an snRNP complex, which renders it
inactive. However, when it is activated, it leaves this complex
and phosphorylates DSIF and NELF, both of which are
negative elongation factors normally bound together with pol

II to stall transcription. This phosphorylation ejects both DSIF

and NELF from the pre-initiation complex, unpausing pol II
and allowing transcription to proceed. Furthermore, P-TEFb
is a CDK that phosphorylates serine 2, marking transcription
elongation and allowing for recruitment of necessary factors
Many other factors necessary for activation, such as
chromatin remodeling complexes, histone acetylases, etc.
G-quadraplexes formation of these unique tertiary structures in
promoter regions can occlude activating proteins, preventing
transcription of the associated gene. If this is true, however, Gquadraplex formation also promotes transcription if it forms from the
non-coding strand, as that would force the promoter of the coding
strand to be exposed an easier target for activating factors.

[Eukaryotes] RNA Polymerase I & III dependent transcription

o Significantly less complex initiation machinery, most likely because these
genes are constantly being expressed. It makes less sense to institute
extensive barriers to activation.

Post-transcriptional modifications: 5 Capping

o Stabilizes mRNA, directs initial splicing, and aids in translation
o Capping reaction three steps:
Dephosphorylation (cleaving - bond) of the 5 end of the nascent
RNA by a phosphatase
Addition of a GTP (GMP added & PPi released) in a 5 to 5 linkage
by guanylyl transferase
Methylation of the guanine by methylase, resulting in a positively
charged guanine. This positively charged guanine can then be
recognized by translation factors (i.e. eIF4E). The translation
factors/mRNA complex can then be recognized by the ribosome to
begin translation
o How does the guanine cap protect the mRNA from degradation?
Normally, exonucleolytic degradation occurs from the 5 end.
However, because the guanine cap is added in a 5 to 5 linkage,
there is a 3 OH presented, which tricks 5 3 exonucleases into
thinking that the 5 end is actually the 3 end. Additionally, the

complex of the 5 cap with translation factors physically prevents

exonucleases from digesting the mRNA.
o How do the capping enzymes actually find the mRNA?
Capping enzymes bind to phosphorylated residues on the CTD,
localizing them to the site of transcription

Post-transcriptional modifications: (alternative) splicing

o Alternative splicing allows for expression of many different protein
isoforms from a single genetic sequence by inclusion of different
permutations of exons
o Mechanism of splicing (refer to Lecture 3, slide 18)
Splicing reactions are carried out via a massive complex called the
spliceosome, which is constituted by ~100 proteins and 5 small
RNA/protein complexes U1, U2, U4, U5, and U6.
U1 first binds and recognizes the 5 splice site via a conserved
sequence (GURAGU) as the beginning of the intron. Branch point
binding protein (BBP) and U2AF bind to a conserved sequence
(UACUAAC). U2 then comes, displaces these two proteins, and
binds to the same sequence. U2 is complementary to the above
sequence for all but the last A, forcing that A to rotate away from the
others, leaving its 2 OH exposed and, upon activation, ready for
nucleophilic attack.
The exposed 2OH near the 3 splice site ultimately attacks the 5
splice site to catalyze the splicing event. However, it is quite far
away how is it brought closer to allow for attack? The U4/U6+U5
complex (U4 binds tightly to, and is a chaperone for, U6, which has
catalytic activity, while U5 helps to stabilize their interaction) helps
by binding the 5 splice site and U2, which is bound at the branch
point with the exposed A. This brings the exposed A closer and
allows its 2 OH hydroxyl to attack at the splice site, creating a lariat
structure, connected to the pre-mRNA by at the 3 splice site.
U6 then attacks the 3 splice site to liberate the lariat completely
(which is debranched by Dbr1 and subsequently degraded by
Xrn2), allowing for joining of the exons.
Post-transcriptional modifications: poly-adenylation
o Two step process, involving cleavage near the 3 end and addition there of
a long tracts of A nucleotides (i.e. poly A sequence)

o Process: Via phosphorylated residues, the CTD recruits several polyadenylation-related factors. These bind to phosphorylated serine 2, which
is a marker of transcription elongation. One of these is CPSF, which binds
to a conserved sequence (usually AAUAAA) slightly upstream of the 3
cleavage site. In complex with CstF, CPSF catalyzes the cleavage event
allowing for addition of poly-A to the 3 end by poly-A polymerase (PAP).
o While PAP is adding A nucleotides to the 3 end, transcription is
continuing. Both the end of polyadenylation and termination are coupled
events, requiring interaction of pol II with termination factors, and a 5 to 3
exonuclease (i.e. Xrn2) to allow for 5 capping.

*Perhaps an important note, and something to think about, is the role the CTD
plays in not only allowing for these events to take place, but in regulating the
order in which they occur. Phosphorylation of specific residues occurs
sequentially by different kinases as they appear in each step of mRNA
transcription and processing.