Transcript 1. Genes and RNA

1. Genes and RNA
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The initial products of all genes is a sequence of ribonucleic acid
(RNA).
RNA is produced by a process that copies the nucleotide sequence in
DNA. Since this process is reminiscent of transcribing (copying)
written words, the synthesis of RNA is called transcription.
The DNA is said to be transcribed into RNA, and the RNA is called
a transcript.
One way to think about the different biological roles of DNA and
RNA is to consider that the DNA (that is, the genome) is the
instruction manual for producing all the RNAs that the cell needs,
whereas RNA is the erasable readout of those parts of the manual
relevant to any given task.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
2. Properties of RNA
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Although RNA and DNA are both nucleic acids, RNA
differs in several important ways:
1. RNA is a single-stranded nucleotide chain, not a double helix. One
consequence of this is that RNA can form a much greater variety of complex
three-dimensional molecular shapes than can double-stranded DNA.
2. RNA has ribose sugar in its nucleotides, rather than deoxyribose. As the
names suggest, the two sugars differ in the presence or absence of just one
oxygen atom. Analogous to the individual strands of DNA, there is a
phosphate-ribose backbone to RNA, with a base covalently linked to the 1
position on each ribose.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
3. Uracil instead of thymine
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The nucleotides of RNA carry the bases
adenine, guanine, and cytosine, but the
pyrimidine base uracil (abbreviated U) is
found in place of thymine:
However,
uracil forms
hydrogen bonds with
adenine just as thymine does.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
4. Classes of RNA
 RNAs
can be grouped into two general
classes:
Some RNAs are intermediaries in the process of
decoding genes into polypeptide chains; these molecules
are called "informational" RNAs.
 In the other class, the RNA itself is the final, functional
product. These RNAs are called "functional" RNAs
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Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
5. Informational RNAs
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For the vast majority of genes, the RNA is only an intermediate in
the synthesis of the ultimate functional product, which is a protein.
The informational RNA of this vast majority of genes is always
messenger RNA (mRNA).
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In prokaryotes, the transcript, as it is synthesized directly from the DNA (the primary
transcript), is the mRNA. In eukaryotes, however, the primary transcript is processed
through modification of the 5’ and 3’ ends and removal of pieces of the primary transcript
(introns). At the end of this pre-mRNA processing, an mRNA is produced.
The sequence of nucleotides in mRNA is converted into the sequence
of amino acids in a polypeptide chain by a process called translation.
In this connection the word translation is used in much the same way
as we use it in translating a foreign language: the cell has a way of
translating the language of RNA into the language of polypeptides.
Proteins are made up of one or more polypeptide chains.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
6. Functional RNAs
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Functional RNAs action is purely at the level of the RNA; they are
never translated into polypeptides. Each class of functional RNA is
encoded by a relatively small number of genes (a few tens to a few
hundred). The main classes of functional RNAs contribute to various
steps in the informational processing of DNA to protein. Two classes
of functional RNAs are found in all organisms:
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Transfer RNA (tRNA) molecules act as transporters that bring amino acids to the
mRNA during the process of translation (protein synthesis). The tRNAs are general
components of the translation machinery; they can bring amino acids to the mRNA of
any protein-coding gene.
Ribosomal RNAs (rRNAs) are components of ribosomes, which are large
macromolecular assemblies that act as guides to coordinate the assembly of the amino
acid chain of a protein. Ribosomes are composed of several types of rRNA and about 100
different proteins. As in the case of tRNA, the rRNAs are general translational
components that can be used to translate the mRNA of any protein-coding gene.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
7. One DNA strand is the template
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Transcription relies on the complementary pairing of bases. The two
strands of the DNA double helix separate locally, and one of the
separated strands acts as a template (alignment guide) for RNA
synthesis. In the chromosome overall, both DNA strands are used as
templates, but in any one gene only one strand is used, and in that
gene it is always the same strand.
One or the other DNA strand is used as transcriptional template.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
8. 5’3’
RNA growth is always in the 5’3’ direction; in other words, nucleotides are always
added at a 3’ growing tip:
RNA polymerase moves always from the 3’ end of the template strand, creating an
RNA strand that grows in a 5’3’ direction (since it must be antiparallel to the
template strand). Some genes are transcribed from one strand of the DNA double helix;
other genes use the other strand as the template
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
9. Transcription in action
Transcription of ribosomal RNA (rRNA) genes in
the developing egg cell of the spotted newt
Eukaryotes have several hundred
identical genes encoding
ribosomal RNA. The long
filaments are DNA molecules
coated with proteins. The fibers
extending in clusters from the main
axes are molecules of ribosomal
RNA which will be used in the
construction of the cell's ribosomes.
Transcription begins at one end of
each gene, with the RNA molecules
getting longer as they proceed
toward completion. Note the large
number (up to 100) of RNA
molecules that are transcribed
simultaneously from each gene.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
10. RNA Polymerases
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In most prokaryotes, a single RNA polymerase does the job of
transcribing all types of RNA.
Eukaryotes have three different RNA polymerases, which specialize
as follows:
1. RNA polymerase I (Pol I) transcribes rRNA genes.
2. RNA polymerase II (Pol II) transcribes protein-coding genes.
3. RNA polymerase III (Pol III) transcribes other functional RNA genes (for example, tRNA
genes).
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In eukaryotes, transcription of nuclear chromosomes takes place
entirely within the nucleus, and the transcripts then move through
nuclear pores out into the cytoplasm, where translation occurs.
Since prokaryotes have no nucleus, there is no comparable movement
of transcripts, and translation can take place immediately, right on the
growing transcript.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
11. Three stages of transcription
 Transcription
is usually described in
terms of three distinct stages:
Initiation
 Elongation
 Termination
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Genetica per Scienze Naturali
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12. INITIATION
A DNA sequence to which RNA polymerase binds to initiate
transcription is termed a promoter.
A promoter is part of the regulatory region adjacent to the coding region of a
gene. Since an RNA transcript is made in the 5’3’ direction, the convention is
to view the gene in the 5’3’ orientation, too (the orientation of the
nontemplate strand), even though transcription actually starts at the 3’ end of the
template strand. By convention the first-transcribed end of the gene is called the
5’ end. Using this view, the promoter is at the beginning of the gene and, so, is
said to be at the 5’ end of the gene, and the regulatory region is called the 5’
regulatory region
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
13. The promoter
Promoter sites have regions of similar sequences, as indicated by the
yellow region in the 13 different promoter sequences in E. coli. Spaces
(dots) included to maximize homology at consensus sequences. The gene
governed by each promoter sequence is indicated on the left. Numbering
is given in terms of the number of bases before () or after (+) the RNA
synthesis initiation point.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
14. The TATA box
Two regions of partial similarity appear in virtually all promoters.
These regions have been termed the -35 (minus 35) and -10 regions
because of their locations relative to the transcription initiation point.
RNA polymerase scans the DNA for a promoter sequence, binds to the
DNA at that point, then unwinds it and begins the synthesis of an RNA
molecule at the transcriptional initiation site. Hence, we see that the
principle of DNA binding is a result of interactions between the
protein (here, the RNA polymerase) and a specific base sequence in
the DNA.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
15. RNA polymerase in bacteria
Schematic diagram of prokaryotic RNA polymerase. The
core enzyme contains two a polypeptides, one b polypeptide,
and one b’ polypeptide. The addition of the s subunit allows
initiation at promoter sites.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
16. The s factor
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In order to recognize their promoters, bacterial RNA polymerase enzymes require a
specialized subunit called the sigma factor (σ), which directly contacts the promoter
sequence. The complex formed by the sigma subunit with the remaining polymerase core
subunits constitutes the bacterial holoenzyme.
Bacteria contain a variety of sigma factors that specifically recognize different promoter
sequences. It is therefore the sigma factor that determines which genes are transcribed.
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All cells have a primary sigma factor, which
directs transcription from the promoters of
essential housekeeping genes, and a variable
number of alternative sigma factors whose
levels or activities are increased in response
to specific signals. E. coli, a symbiotic
bacterium leading a relatively sheltered life in
the gut of other organisms, has only 7 sigma
factors.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini
17. Structure of a bacterial RNA polymerase
The structure of the T. aquaticus
holoenzyme shows how three structural
domains of the sigma subunit bind to the
core enzyme in a position to recognize the
promoter elements. The DNA is
numbered relative to the transcription start
site at +1. The σ2 domain recognizes the 10 region (red), while the σ3 domain binds
to the flanking base pairs of the extended
-10 region. The σ4 domain, which binds to
the -35 element (red), is anchored to a
flexible flap of the β subunit that may
allow movement of the σ4 subunit to
allow for different spacings between the
-35 and -10 regions.
Genetica per Scienze Naturali
a.a. 05-06 prof S. Presciuttini