Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 1. Genes and RNA The initial products...

Genetica per Scienze Naturalia.a. 05-06 prof S. Presciuttini

1. Genes and RNA TThe initial products of all genes he initial products of all genes is a sequence of is a sequence of ribonucleic acid ribonucleic acid

(RNA).(RNA). RNA is produced by a process that copies the nucleotide sequence in RNA is produced by a process that copies the nucleotide sequence in

DNA. Since this process is reminiscent of transcribing (copying) DNA. Since this process is reminiscent of transcribing (copying) written words, the synthesis of RNA is called written words, the synthesis of RNA is called transcriptiontranscription..

The DNA is said to be transcribed into RNA, and the RNA is called The DNA is said to be transcribed into RNA, and the RNA is called a a transcripttranscript..

One way to think about the different biological roles of DNA and 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 RNA is to consider that the DNA (that is, the genome) is the instruction manual for producing all the RNAs that the cell needs, instruction manual for producing all the RNAs that the cell needs, whereas RNA is the erasable readout of those parts of the manual whereas RNA is the erasable readout of those parts of the manual relevant to any given task. relevant to any given task.


2. Properties of RNA Although RNA and DNA are both nucleic acids, RNA Although RNA and DNA are both nucleic acids, RNA

differs in several important ways:differs in several important ways:1. RNA is a single-stranded nucleotide chain, 1. RNA is a single-stranded nucleotide chain, not a double helixnot a double helix. One . One

consequence of this is that RNA can form a much greater variety of consequence of this is that RNA can form a much greater variety of complex three-dimensional molecular shapes than can double-stranded complex three-dimensional molecular shapes than can double-stranded DNA. DNA.

2. RNA has 2. RNA has riboseribose sugar in its nucleotides, rather than deoxyribose. As the sugar in its nucleotides, rather than deoxyribose. As the names suggest, the two sugars differ in the presence or absence of just one 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 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 phosphate-ribose backbone to RNA, with a base covalently linked to the 1 position on each ribose.position on each ribose.


3. Uracil instead of thymine The The nucleotides nucleotides of of RNA carry the bases RNA carry the bases

adenine, guanine, and cytosine, but the adenine, guanine, and cytosine, but the pyrimidine base pyrimidine base uraciluracil (abbreviated U) is (abbreviated U) is found in place of thyminefound in place of thymine::

However, uracil forms However, uracil forms hydrogen bonds with hydrogen bonds with adenineadenine just as thymine does. just as thymine does.


4. Classes of RNA RNAs can be grouped into two general RNAs can be grouped into two general

classesclasses:: Some RNAs are intermediaries in the process of Some RNAs are intermediaries in the process of

decoding genes into polypeptide chains; these molecules decoding genes into polypeptide chains; these molecules are calledare called " "informationalinformational" RNAs." RNAs.

In the other class, the RNA itself is the final, functional In the other class, the RNA itself is the final, functional product. product. TThese RNAs hese RNAs are calledare called " "functionalfunctional" RNAs" RNAs


5. Informational RNAs

For the For the vast majority of genesvast majority of genes, the RNA is only an , the RNA is only an intermediateintermediate in in the synthesis of the ultimate functional product, which is a protein. the synthesis of the ultimate functional product, which is a protein. The informational RNA of this vast majority of genes is always The informational RNA of this vast majority of genes is always messenger RNA (messenger RNA (mRNAmRNA).). In prokaryotes, In prokaryotes, the transcriptthe transcript, as it is synthesized directly from the DNA (the primary , as it is synthesized directly from the DNA (the primary

transcript), transcript), is the mRNAis the mRNA. In eukaryotes, however, the . In eukaryotes, however, the primary transcriptprimary transcript is processed is processed through modification of the 5through modification of the 5’’ and 3 and 3’’ ends and removal of pieces of the primary ends and removal of pieces of the primary transcript (introns). At the end of this pre-mRNA processing, an mRNA is produced. 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 The sequence of nucleotides in mRNA is converted into the sequence of amino acids in a polypeptide chain by a process called of amino acids in a polypeptide chain by a process called translationtranslation. . In this connection the word translation is used in much the same way 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 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. translating the language of RNA into the language of polypeptides. Proteins are made up of one or more polypeptide chains.Proteins are made up of one or more polypeptide chains.


6. Functional RNAs FFunctional RNAs action is purely at the level of the RNA; they are unctional RNAs action is purely at the level of the RNA; they are

never translated into polypeptides. Each class of functional RNA is never translated into polypeptides. Each class of functional RNA is encoded by a relatively small number of genes (a few tens to a few encoded by a relatively small number of genes (a few tens to a few hundred).hundred). The main classes of functional RNAs contribute to various The main classes of functional RNAs contribute to various steps in the informational processing of DNA to protein. Two classes steps in the informational processing of DNA to protein. Two classes of functional RNAs are found in all organisms:of functional RNAs are found in all organisms: Transfer RNA (Transfer RNA (tRNAtRNA) molecules act as transporters that ) molecules act as transporters that bring amino acids to the bring amino acids to the

mRNAmRNA during the process of translation (protein synthesis). The tRNAs are general 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 components of the translation machinery; they can bring amino acids to the mRNA of any protein-coding gene.any protein-coding gene.

Ribosomal RNAs (Ribosomal RNAs (rRNAsrRNAs) are components of ) are components of ribosomesribosomes, which are large , which are large macromolecular assemblies that act as guides to coordinate the assembly of the amino 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 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 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.components that can be used to translate the mRNA of any protein-coding gene.


7. One DNA strand is the template Transcription relies on the complementary pairing of bases. The two Transcription relies on the complementary pairing of bases. The two

strands of the DNA double helix separate locally, and one of the strands of the DNA double helix separate locally, and one of the separated strands acts as a separated strands acts as a templatetemplate (alignment guide) for RNA (alignment guide) for RNA synthesis. In the chromosome overall, both DNA strands are used as synthesis. In the chromosome overall, both DNA strands are used as templates, but in any one gene templates, but in any one gene only one strandonly one strand is used, and in that is used, and in that gene it is always the same strand.gene it is always the same strand.

One or the other DNA strand is used as transcriptional template.


8. 5’3’RNA growth is always in the 5RNA growth is always in the 5’’33’’ direction; in other words, direction; in other words, nucleotides are always nucleotides are always added at a 3added at a 3’’ growing tip growing tip::

RNA polymerase moves RNA polymerase moves always always from the 3from the 3’’ end of the template strand, creating an end of the template strand, creating an RNA strand that grows in a 5RNA strand that grows in a 5’’33’’ direction (since it must be direction (since it must be antiparallelantiparallel to the to the template strand). template strand). SSome genes are transcribed from one strand of the DNA double helix; ome genes are transcribed from one strand of the DNA double helix; other genes use the other strand as the templateother genes use the other strand as the template


9. Transcription in actionEukaryotes have Eukaryotes have several hundred several hundred identical genes encoding identical genes encoding ribosomal RNAribosomal RNA. The long . The long filaments are DNA molecules filaments are DNA molecules coated with proteins. The fibers coated with proteins. The fibers extending in clusters from the main extending in clusters from the main axes are molecules of ribosomal axes are molecules of ribosomal RNA which will be used in the RNA which will be used in the construction of the cell's ribosomes. construction of the cell's ribosomes. TTranscription begins at one end of ranscription begins at one end of each gene, with the RNA molecules each gene, with the RNA molecules getting longer as they proceed getting longer as they proceed toward completion. Note the large toward completion. Note the large number (up to 100) of RNA number (up to 100) of RNA molecules that are transcribed molecules that are transcribed simultaneously from each gene.simultaneously from each gene.

TTranscription of ranscription of ribosomal RNA (rRNA) genesribosomal RNA (rRNA) genes in in the developing egg cell of the spotted newtthe developing egg cell of the spotted newt


10. RNA Polymerases In most prokaryotes, a single RNA polymerase does the job of In most prokaryotes, a single RNA polymerase does the job of

transcribing all types of RNA.transcribing all types of RNA. EEukaryotes have three different RNA polymerases, which specialize ukaryotes have three different RNA polymerases, which specialize

as follows:as follows:1. RNA polymerase I (Pol I) transcribes 1. RNA polymerase I (Pol I) transcribes rRNA genesrRNA genes..

2. RNA polymerase II (Pol II) transcribes 2. RNA polymerase II (Pol II) transcribes protein-coding genesprotein-coding genes..

3. RNA polymerase III (Pol III) transcribes 3. RNA polymerase III (Pol III) transcribes other functional RNA genesother functional RNA genes (for example, tRNA (for example, tRNA genes).genes).

IIn eukaryotesn eukaryotes,, transcription of nuclear chromosomes takes place transcription of nuclear chromosomes takes place entirely entirely within the nucleuswithin the nucleus, and the transcripts then move through , and the transcripts then move through nuclear poresnuclear pores out into the cytoplasm, where out into the cytoplasm, where translationtranslation occurs. occurs. Since prokaryotes have no nucleus, there is no comparable movement Since prokaryotes have no nucleus, there is no comparable movement of transcripts, and translation can take place immediately, right on the of transcripts, and translation can take place immediately, right on the growing transcript.growing transcript.


11. Three stages of transcription TTranscription ranscription is usually described in is usually described in

terms of terms of three distinct stages:three distinct stages:InitiationInitiationElongationElongationTTerminationermination


12. INITIATION

A DNA sequence to which RNA polymerase binds to initiate A DNA sequence to which RNA polymerase binds to initiate transcription is termed a transcription is termed a promoterpromoter..

A promoter is part of the A promoter is part of the regulatory regionregulatory region adjacent to the coding region of a adjacent to the coding region of a gene. gene. SSince an RNA transcript is made in the 5ince an RNA transcript is made in the 5’’33’’ direction, the convention is direction, the convention is to view the gene in the 5to view the gene in the 5’’33’’ orientation, too (the orientation of the orientation, too (the orientation of the nontemplate strand), even though transcription actually starts at the 3nontemplate strand), even though transcription actually starts at the 3’’ end of end of the template strand. By convention the first-transcribed end of the gene is called the template strand. By convention the first-transcribed end of the gene is called the 5the 5’’ end. Using this view, the promoter is at the beginning of the gene and, so, end. Using this view, the promoter is at the beginning of the gene and, so, is said to be at the 5is said to be at the 5’’ end of the gene, and the regulatory region is called the 5 end of the gene, and the regulatory region is called the 5’’ regulatory regionregulatory region


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.


14. The TATA boxTwo regions of partial similarity appear in virtually Two regions of partial similarity appear in virtually all promotersall promoters..

These regions have been termed the These regions have been termed the --35 (minus 35) and 35 (minus 35) and --10 regions 10 regions because of their locations relative to the transcription initiation point.because of their locations relative to the transcription initiation point.

RNA polymerase scans the DNA for a promoter sequence, binds to the 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 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 molecule at the transcriptional initiation site. Hence, we see that the principle of DNA binding is a result of interactions between the principle of DNA binding is a result of interactions between the protein (here, the RNA polymerase) and a specific base sequence in protein (here, the RNA polymerase) and a specific base sequence in the DNAthe DNA..


15. RNA polymerase in bacteria

Schematic diagram of prokaryotic RNA polymerase. The core enzyme contains two polypeptides, one polypeptide, and one ’ polypeptide. The addition of the subunit allows initiation at promoter sites.


16. The factor In order to recognize their promoters, bacterial RNA polymerase enzymes require a In order to recognize their promoters, bacterial RNA polymerase enzymes require a

specialized subunit called the sigma factor (σ), which specialized subunit called the sigma factor (σ), which directly contacts the promoter directly contacts the promoter sequencesequence. The complex formed by the sigma subunit with the remaining polymerase core . The complex formed by the sigma subunit with the remaining polymerase core subunits constitutes the bacterial subunits constitutes the bacterial holoenzymeholoenzyme..

Bacteria contain a variety of sigma factors that specifically recognize different promoter 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.sequences. It is therefore the sigma factor that determines which genes are transcribed.

All cells have a All cells have a primary sigma factorprimary sigma factor, which , which directs transcription from the promoters of directs transcription from the promoters of essential housekeeping genes, and a variable essential housekeeping genes, and a variable number of alternative sigma factors whose number of alternative sigma factors whose levels or activities are increased levels or activities are increased in response in response to specific signalsto specific signals. E. coli, a symbiotic . E. coli, a symbiotic bacterium leading a relatively sheltered life in bacterium leading a relatively sheltered life in the gut of other organisms, has only the gut of other organisms, has only 77 sigma sigma factors.factors.


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 1. Genes and RNA The initial products...

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