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UNIVERSITY OF OSLO
Faculty of Mathematics and Natural Sciences
Exam in:
MBV2010 Molecular Biology
Day of exam:
June 6, 2011
Exam hours: 9:00-12:00 (3 hours)
This examination paper consists of 2 pages.
Appendices: None
Permitted materials: None
Make sure that your copy of this examination paper is complete before answering.
Numbers in brackets indicate the maximum number of points for each question. The maximum
number of points for the entire exam is 100.
1. In which molecular processes are the following proteins involved?
DNA polymerase V - DNA repair (replication in SOS response) p. 532
Polynucleotide phosphorylase (PNPase) - RNA processing (degradation) p. 347
Aminoacyl-tRNA synthetase - translation p. 388
Primase - replication p. 481-482
Integrase - site-specific recombination p. 551
Initiation factor 2 (IF-2) - translation p. 396-397
TATA-binding protein (TBF) - transcription p. 313-314
Proliferating cell nuclear antigen (PCNA) - replication p. 480
MAP kinase - signal transduction (regulation of gene expression) p. 434
cI repressor - (regulation of) transcription p. 445
DNA glycosylases - DNA repair p. 526
MutS - DNA repair (mismatch) p. 529-530
Catabolite activator protein (CAP) - (regulation of) transcription p. 430-431
DNA gyrase - replication, transcription, recombination, supercoiling p. 472-473
SR proteins - RNA processing (splicing) p. 358-359
(15)
2. Define briefly (1-5 sentences) the following terms:
a) Stringent response - a response in bacteria to lack of nutrients. If bacteria cannot
grow due to low levels of nutrients, transcription of genes (particularly tRNA and
ribosomal RNA genes) is reduced to a few percent of the normal level. The response is
mediated by the DksA protein and alarmones (ppGpp and pppGpp). p. 343
b) SOS response - a response in bacteria to extensive DNA damage. In this response
the damaged DNA region is bypassed during replication by DNA polymerase V
1
which synthesizes DNA in the absence of correct base pairing to the template strand.
p. 531-532
c) Alternative splicing - alternative joining of different exons to produce more than
one variant of a specific mRNA. p. 359-362
d) Chromosome painting - visualization of chromosome territories by labeling
different regions of a chromosome with the same fluorescent dye. p. 274
e) Attenuation - premature termination of transcription in bacteria, primarily found
in transcription of operons for amino acid synthesis. p. 340
3.
(15)
a) Describe the process of splicing of GU-AG introns.
- splicing of GU-AG introns is catalyzed by spliceosomes, small ribonucleoprotein
complexes (snRNPs) that bind to specific sites in introns, cleaving and releasing the
intron sequences and joining the exons. There are five snRNPs (U1, U2, U4, U5, and
U6), plus a few auxiliary proteins, involved in intron splicing.
The process starts with cleavage of the 5’ splice site by a transesterification reaction
that links the 5’ end of the intron to a specific adenine nucleotide in the intron
sequence. A second transesterification reaction links the 5’ phosphate of the
downstream exon to the free 3’-OH group at the 5’ splice site, thereby releasing the
intron sequence. In most cases, released introns are degraded but in some cases a
portion of an intron remains intact and functions in other processes, e.g. as small
nucleolar RNA. p. 356-358
(15)
b) How can splicing of GU-AG introns be regulated?
By proteins that are involved in selecting the splice sites, e.g. SR proteins in
conjunction with exonic splicing enhancers and exonic splicing silencers. p. 358-359
(5)
c) What other types of introns are known?
(5)
- AU-AC introns that also use spliceosomes for splicing.
- Group I, II, and III introns, mostly found in organelles. These are all self-splicing introns.
- Composite introns in which introns occur in introns. They follow a defined order
of splicing.
- Pre-tRNA introns and introns in archaebacteria that are removed by ribonucleases.
p. 355 and 367
4.
d) List ways, other than splicing, whereby RNAs can be processed in cells.
- end modifications (5’-cap, 3’ polyA-tail) p. 349-352
- cutting (release of ribosomal and tRNAs) p. 344-345
- chemical modifications (e.g. methylations and base modifications in tRNA) p. 346
- editing (special type of chemical modification that alters a triplet codon in a coding
region)
p. 369-371
- degradation (not really processing in a strict sense) p. 346-347 and 371-375
(10)
a) Describe the process of translation in bacteria.
(15)
Translation in bacteria begins with binding of the small ribosomal subunit (with
initiation factor IF-3) to the ribosome binding site (also called Shine-Dalgarno
sequence) of the mRNA. After binding of the small ribosomal subunit, the initiator
tRNA (loaded with formyl-methionine) and the large ribosomal subunit are recruited to
2
the complex, aided by initiation factors IF-2 and IF-1, respectively. Assembly of the
complex forms three sites, an aminoacyl (A) site, peptidyl (P) site, and exit (E) site.
Initially, the ribosome and mRNA are aligned such that the initiator tRNA is
positioned in the peptidyl site by codon-anticodon interactions.
In the elongation phase of translation tRNAs, charged with amino acids, enter the
complex through the A site, mediated by elongation factor 1A (EF-1A). Formation of
the peptide bond between the C-terminal amino acid in the P site and the amino acid in
the A site is catalyzed by a peptidyl transferase. This activity is probably associated
with the 23S ribosomal RNA (ribozyme activity). After peptide bond formation the
ribosome translocates to the next codon on the mRNA, aided by elongation factor 2
(EF-2). Translocation moves the uncharged tRNA from the P site to the E site from
which it is expelled.
Translation terminates when one of the translation stop codons in the mRNA (UAA,
UAG, UGA) is in the A site. Only release factors (RF-1 or RF-2) that enter the A site
can interact with translation stop codons and release the newly synthesized peptide in
an energy-requiring reaction. Dissociation of the ribosome into separate large and
small subunits is mediated by a ribosome recycling factor.
p. 395-405
b) How does translation in bacteria differ from translation in eukaryotes?
Almost no difference in elongation and termination except that there is no exit site
formed in eukaryotic ribosomes and that eukaryotes have less elongation and release
factors and probably no ribosome recycling factor.
The initiation process is different in eukaryotes: a preinitiation complex, consisting of
the small ribosomal subunit and initiator tRNA (plus initiation factors) is formed that
binds to the 5’ cap of eukaryotic mRNAs. The complex scans the mRNA in 5’ -> 3’
direction until it identifies the translation start codon (the codon is embedded into the
Kozak consensus sequence). The large ribosomal subunit is recruited and translation
initiates. More initiation factors than in bacteria are involved in the initiation process.
There is probably also a role of the polyA tail in translation initiation.
Some eukaryotic (and viral) mRNAs have an internal ribosome entry site (IRES)
whose function is similar to the bacterial ribosome binding site, allowing capindependent initiation of translation.
p. 397-399
(10)
c) How can translation be regulated?
Global regulation by phosphorylation of initiation factor 2 in eukaryotes (eIF-2)
inhibits translation.
Transcript-specific regulation by reversible binding of proteins to the 5’ untranslated
regions (5’ UTRs) of mRNAs thereby blocking initiation of translation.
p. 399-400
(5)
d) What is known about degradation of proteins?
Specific proteases degrade proteins in bacteria. In eukaryotes most proteins seem to be
degraded by proteasomes. In the latter case proteins are targeted for degradation by
ubiquitinylation. Proteasomes are large multisubunit cylinder-like complexes. Proteins
enter the complex unfolded and are degraded into small oligopeptides that are further
degraded in the cytoplasm into individual amino acids.
p. 414-415
(5)
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