Transcript 1. ELONGATION

1. ELONGATION
Shortly after initiating transcription, the sigma factor dissociates from the RNA polymerase,
which moves along the DNA, maintaining a transcription "bubble" to expose the template strand,
and catalyzes the 3’ elongation of the RNA strand. The polymerase compares free ribonucleotide
triphosphates with the next exposed base on the DNA template and, if there is a complementary
match, adds it to the chain.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
2. TERMINATION
Specific nucleotide sequences in the DNA act as signals for
RNA chain termination. In the mechanism called direct
termination, the termination signal consist of about 40 bp
containing a GC-rich palindrome, followed by an oligo A
region, which forms a local stem-loop structure in the RNA.
The resulting double-stranded RNA section is called a
hairpin loop. It is followed by the terminal run of U's that
correspond to the A residues on the DNA template. This
sequence disrupts the base pairing of newly synthesized
RNA with the DNA template, forcing the RNA and the
polymerase to fall off.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
3. RNA Processing in Eukaryotes
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Transcription works in much the same way in eukaryotes as in prokaryotes; that is,
there are specific promoter sequences to which the RNA polymerase binds, and the
polymerase moves along the gene synthesizing RNA in the 5’3’ direction.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
4. The RNA polymerase II holoenzyme
Model of RNA Polymerase II
Transcription Initiation
Machinery.The machinery
depicted here encompasses over
85 polypeptides in 10 (sub)
complexes: core RNA polymerase
II (RNAPII) consists of 12
subunits; TFIIH, 9 subunits;
TFIIE, 2 subunits; TFIIF, 3
subunits; TFIIB, 1 subunit, TFIID,
14 subunits; core SRB/mediator,
more than 16 subunits; Swi/Snf
complex, 11 subunits; Srb10
kinase complex, 4 subunits; and
SAGA, 13 subunits.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
5. mRNA maturation
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In eukaryotes, the initial product of transcription, the primary RNA transcript, is
processed in several ways before its transport to the cytosol. These processing steps
are all performed by specific proteins that bind to the RNA. Until it reaches its final,
mature form, the primary transcript is sometimes called pre-mRNA.
First, during transcription, a cap consisting of a 7-methylguanosine residue is
added to the 5’ end of the transcript, linked by a triphosphate bond.
Then an AAUAAA sequence near the 3’ end is recognized by an enzyme that cuts
off the end of the RNA approximately 20 bases farther down. At this time a stretch
of 150 to 200 adenine nucleotides called a poly(A) tail is added at the cut 3’ end.
Next, a crucial splicing step removes any introns from the RNA transcript,
converting pre-mRNA into mature mRNA.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
6. Mechanism of exon splicing
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For many eukaryotic genes the capped and tailed transcripts are shortened by the
elimination of internal introns before transport into the cytoplasm.
Exon-intron junctions of mRNAs show specific sequences that are highly conserved, i.e., they
are the same in most introns in most species. There is a GU at the 5’ splice site of the intron
and an AG at the 3’ splice site in virtually all cases examined ("the GU-AG rule")
Consensus sequences of 5’ and 3’ splice
junctions in eukaryotic mRNAs. Almost
all introns begin with GU and end with
AG. From the analysis of many exon
intron boundaries, extended consensus
sequences of preferred nucleotides at
the 5’ and 3’ ends have been
established. In addition to AG, other
nucleotides just upstream of the 3 splice
junction also are important for precise
splicing.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
7. The spliceosome
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Other less well conserved sequences are found flanking these. These common
configurations of the pre-mRNA are recognized by small nuclear
ribonucleoprotein particles, or snRNPs, which catalyze the cutting and splicing
reactions.
During the process of splicing,
the snRNPs, the primary
transcript, and associated
factors all come together to
form a high-molecular-weight
(60S) ribonucleoprotein
complex, called a
spliceosome, which catalyzes
the splicing reactions.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
8. The translation
At INITIATION, the ribosome
recognizes the starting point in a
segment of mRNA and binds a
molecule of tRNA bearing a single
amino acid. In all bacterial proteins,
this first amino acid is Nformylmethionine.
In ELONGATION, a second amino
acid is linked to the first one. The
ribosome then shifts its position on
the mRNA molecule, and the
elongation cycle is repeated.
At TERMINATION, when the stop
codon is reached, the chain of amino
acids folds spontaneously to form a
protein. Subsequently, the ribosome
splits into its two subunits, which
rejoin before a new segment of mRNA
is translated.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
9. The ribosome
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
10. Polyribosomes
Clusters of ribosomes may sit on a mRNA and make proteins, each
making a strand of polypeptides. These clusters are called
polyribosomes. When they are free in the cytoplasm, they are called free
polyribosomes (linked by the mRNA). Or, they may bind to rough
endoplasmic reticulum.
Ribosomes are visualized as small (20 X
30 nm) ribonucleoprotein particles. They
are formed from two subunits. The two
ribosomal subunits leave the nucleus
separately through the nuclear pores .
The pores are structured to allow transit
of only the subunits. Whole ribosomes
are formed outside in the cytoplasm. This
prevents protein synthesis from occurring
in the nucleus.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
9. Chain termination
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Three codons of the genetic code, UAG, UGA, and UAA, do not
specify an amino acid. These are called stop codons or termination
codons. They can be regarded as punctuation marks ending the
message encoded in the mRNA. Stop codons often are called
nonsense codons.
The three stop codons are not recognized by a tRNA, but instead by
protein factors called release factors. When the peptidyl-tRNA is in
the P site, the release factors bind to the A site in response to the chain
terminating codons. The polypeptide is then released from the P site,
and the ribosomes dissociate into two subunits, ending translation.
In one way of analyzing DNA sequences to look for potential genes,
computers are programmed to look for open reading frames (ORFs),
which are long DNA sequences beginning with an initiation codon
(for example 5-ATG-3) and ending with one of the three stop codons.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini