| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
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Readings: Brown, 9: 197-198; 207-212
Outline:
A. Basic Features of RNA Processing
1. Prokaryotic:
2-5' half life; polycistronic; seldom modified; used immediately
Coupled transcription and translation; many ribosomes simultaneously
... called polyribosomes or polysomes for short....
[Brown, Fig 9.5]
Polycistronic mRNAs: coordinate expression of >1 gene: operon
Multiple translational start sites
per mRNA
Control of translation by mRNA secondary structure
Ribosomes traversing 1st cistron sometimes open up start codon
for 2nd cistron
Polarity the result of mutations in the 1st cistron: no
translation of 2nd cistron ... good example of Polarity mutations
are those induced by IS
or Transposon insertions
...
Recall: A cistron is a Gene by the genetic definition of the cis-trans complementation test
2. Eukaryotic:
long half life; monocistronic; always modified; transport to cytoplasm
Most post-transcription modifications of the primary transcript occur in nucleus, before transport to cytoplasm ... these modifications convert the transcript (hnRNA) into mRNA
Initial pre-mRNA transcript:
hnRNA ... [Brown, Section 9.1.2]
hnRNA - Heterogeneous
Nuclear RNA ...
Complexed during transcription with ~20 Nuclear Proteins, 6 of
which are the common core proteins which form a bead structure
on the RNA.
The Protein-hnRNA complexes
form Heterogeneous Nuclear Ribonucleoprotein particles: hnRNPs
...
These are the substrates for Capping, poly(A) addition, and Splicing
Splicing can occur without Capping or Poly(A)
Protein complexes facilitate hybridization and stability of hnRNA
3. Major Eukaryotic Modifications
events: ... [Brown, Fig 9.2]
G capping of 5' end of pre-mRNA
Cleavage and Poly(A) addition to 3' end
Splicing out (removal of) Introns
Some methylation of bases
Alternative Splicing: ... [Brown, Fig 9.3 B, Fig 9.16]
control exerted by protein cofactors mainly ... usually tissue-specificity
is seen for different splicing alternatives
a. Methylated G Cap at 5' end: ... [Brown, Fig 9.11]
Guanylyl transferase catalyzes addition of GTP in 5'->5' direction, with release of PPi from the GTP, and release of Pi from the terminal triphosphate of the RNA transcript.
Then one of 3 methyl Capping reactions occurs:
1) Cap 0: a methyl group is added to the G-7
posn: Guanine-7-MethylTransferase
Present in all eukaryotic mRNA ... no further capping in unicelluar
eukaryotes
2) Cap 1: a methyl group is then added to the
2'-OH of the ribose of the 1st nucleotide in the original transcript:
2'-O-MethylTransferase
Present in most eukaryotic mRNA ... if base is A, it too can
be methylated
3) Cap 2: a similar methylation of the 2nd nucleotide
of the original transcript
Present in about 10-15% of total capped population ...
b. Poly(A) on 3' end: ... [Brown, Fig 9.12]
Transcripts are cleaved at
3' end past a highly conserved AAUAAA sequence.
Such Cleavage involves some protein cofactors ... [Brown,
Fig 9.12]
This eliminates the need for precise transcription termination.
The Poly(A) polymerase (PAP) adds ~200 A residues to most mRNAs
Poly(A)-Binding Protein (PABP) binds to the poly(A) stoichiometrically, one protein every 10-20 bases ... increases stability and enhances translation
In recombinant DNA work, the
3'- poly(A) is important for isolation of mRNA:
use of oligo(T) columns ... cDNA cloning: ... [Brown,
Fig 3.11]
Histone mRNAs are poly(A)-free.
c. Internal Methylation: N-6 methylation of Adenines, at frequency
of about 0.1%
4. Eukaryotic RNA Splicing:
Introns and Exons
Eukaryotic genes are
often interrupted genes:
Coding sequence is interrupted by noncoding sequences: ...
[Brown, Box 1.1]
Removal of the Introns in RNA transcript modification is called
RNA splicing;
splicing occurs in the nucleus before transport to the cytoplasm
Exons are Expressed sequences: these sequences are those present in mature mRNA
NOTE: Some of these sequences, notably at 5' and 3' ends, need NOT be translated into protein sequences! ... so Exon sequences are NOT necessarily CODING sequences !!
Introns are InterVening Sequences (IVS), sequences found between Exon sequences in the genomic DNA, i.e. in the gene.
Thus, cDNA clones contain
only Exon sequences;
Genomic clones contain BOTH Exon and Intron sequences
cDNA clones may contain non-coding Exon sequences.
Introns are present in nearly all higher eukaryotic DNA, and usually are much larger than Exons ... this is a real problem for cloning of human or mouse DNA.
Introns are present in very
few genes in lower eukaryotes, e.g. yeast or Dictyostelium
... and not at all in bacteria (1-3 exceptions).
Introns usually do NOT contain genes; mitochondrial DNA introns are an interesting exception, in which a few restriction endonuclease genes are encoded.
Genetics: mutations only in Exons; mutations should be
clustered ...
Distances of mutations in gene and protein very different
Overlapping genes: Two types
1. Same reading frame, but one protein is shorter than other:
homologues
2. Different reading frames, non-homologous proteins, eg phage
phiX174
Alternative Splicing: ... [Brown, Fig 9.3 B, Fig 9.16]
Usual case: a given Exon is present in one mRNA but absent in
another, alternatively spliced mRNA, eg. fly myosin genes, in
different cell types ...
Genes encoded by Introns: site-specific endonucleases
Most are Group I introns ... e.g. omega intron in yeast rRNA
mitochondrion gene
Recognizes an 18 bp site in target, nuclease cleaves, intron
is copied into ds break
5. Editing of RNA: ... [Brown, Fig 9.3 A]
Insertion, deletion, substitution of nucleotides ... changes the
Genetic Information !!
Found in some human genes, eg Apo-B gene in intestine: CAA ->
UAA: stop codon
Extensive use of editing in trypanosome mitochondria ...
and in Leishmania, via use of Guide RNAs ...
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug
Smith