Lecture 5 (2015)Apunte Inglés
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Ingrid Guarro Marzoa
Lecture 5-Transcription and RNA diversity:
The central dogma of molecular biology states that information flows in the
DNAàRNAàprotein sense. All cells from bacteria to humans express their
genetic information in this way. There are some viruses (retroviruses) that don’t
follow this rule and that information flows in RNAàDNA sense. This process
only belongs to RNA viruses and has been the key to fight against retroviruses
such as AIDS. In some other cases, RNA can be copied into RNA in a process
known as autoreplication.
• Transcription: Transcription is the process in which you copy one of the strands of DNA into RNA. This process has some similarities to the process of DNA replication.
Transcription firstly starts with the opening and unwinding of a small portion of the DNA double helix. Then, only one strand will be copied. This strand will act as a template and that’s why we call it the template strand. The RNA chain is determined by the complementary base-pairing of the template strand. The other strand of DNA will be called coding strand because it has the code of the new RNA chain; it’s equivalent to the RNA chain but instead of uracils it has thymines.
The enzymes that perform transcription are called RNA polymerases and as the DNA polymerases, they catalyze the formation of phosphodiester bonds that link the nucleotides together to form a linear chain. All polymerases only add nucleotides on the 3’ and this rule is the same for RNA polymerases.. The new chain is extended by one nucleotide at a time in the 5’à3’ direction such as in DNA and it will be complementary to the template strand. Because new and template strands have to be antiparallel, depending on the direction of the DNA in the gene, the complementary strand will different so the definition of coding strand and template strand will vary depending on the direction in which the gene is transcribed.
2014/2015 Molecular biology Ingrid Guarro Marzoa RNA polymerases don’t need a primer to start the synthesis of the new RNA chain because they do not check the last nucleotide added. Transcription doesn’t need to be as accurate as DNA replication; RNA polymerases make about one mistake every 104 nucleotides copied into RNA and the consequences of an error in RNA transcription are much less significant than that in DNA replication.
The process of transcription done by RNA polymerases is controlled by the places that define the promoter (start point) and terminator (end point). Some other words that we usually use to define these regions are: proximal region (closer to the promoter) and distal region (closer to the terminator) or upstream (closer to the promoter) and downstream (closer to the terminator). These regions are transcriptional unit segments that are going to be transcribed into RNA.
RNA polymerase recognizes the promoter region and starts to copy one strand of DNA into RNA by putting complementary bases. It keeps adding nucleotides at the 3’ end until it finds the terminator. The reaction is almost the same that the one in DNA. The enzyme has a cavity that allows/fits the template strand of DNA and will allow RNA to exit.
We can summarize transcription in 4 steps: 1. Template recognition: RNA polymerase has to recognize the promoter.
The two strands of DNA are going to be separated by a helicase activity.
2. Initiation: the RNA polymerase adds the first nucleotides complementary to the template strand.
3. Elongation: From this point on, the RNA polymerase moves in 5’à3’ direction and elongates the RNA copying information from the template strand. Topoisomerases will prevent the supercoiling of the DNA strands.
2014/2015 Molecular biology Ingrid Guarro Marzoa 4. Termination: the RNA polymerase reads the terminator sequence and stops adding nucleotides.
Most illnesses related to the transcription process occur during template recognition and initiation.
o Promoters in prokaryotes: The sequence of the promoter is special and it is found in -10(TATAAT) and 35(TTGACA), (The numbers are relative to the number of nucleotides upstream from the initiation point). These two sequences are found in most bacterial genes but there’s variability. Both sequences are called consensus sequences because is the more common. In the case of the -10 sequence, the first T is 80% always there and the following A is not present in only 5% of the promoters. From the energetic point of view, the presence of specific bases in the -10 sequence allows a stronger interaction with the RNA polymerase. If many bases are absent of it, the RNApolymerase will not be able to bind (and detect) the promoter sequence and the gene will not be expressed. In a laboratory, if we want high levels of expression we will create a promoter with the same nucleotides of the -10 and -35 sequences and transcription will be more efficient, but if you want to reduce the expression level of a particular gene, you will change the bases so the RNA polymerase will have problems to detect the promoter region and the transcription will be less efficient. The closer is the sequence to the consensus sequence, the more efficient the process is. In prokaryotes, RNA polymerase needs sigma subunits because these subunits are the ones that recognize the promoter.
o Promoters in eukaryotes: In eukaryotes, RNA polymerase recognizes the promoter by many additional proteins called general transcription factors because they can be found in almost all eukaryotic genes. At the promoter we find an important sequence in 20 (TATA). This sequence is present in almost all of our genes but there are also other necessary sequences called enhancers (intensificadors). The proteins that cut these enhancers are called specific transcription factors because they are specific for different tissues. Ex: our liver cells are different because they express a collection of genes that allow them to be hepatocytes.
This is because they use different enhancers. Enhancers are recognized by specific transcription factors that will only operate in those genes having the 2014/2015 Molecular biology Ingrid Guarro Marzoa enhancer sequence. Because of the complexity of eukaryotes, we can find 3 different RNA polymerases depending on the genes that they transcribe: 1. RNA polymerase II: takes care of almost of our genes, synthetizes the precursors of our genes and produces de basal complex.
2. RNA polymerases I and III: produce rRNAs, tRNAs and snRNAs.
The protein that recognizes the TATA sequence is the TFIID. This protein is a complex of polypeptides. Once TFIID is bound to the promoter, it will drive the assembly of the basal complex. The basal complex is not sufficient for traanscrption and needs the work of the specific transcription factors. The contact of this specific transcription factors with the basal complex releases the RNA polymerase to start RNA synthesis.
o Terminator in prokaryotes: At the end of a bacterial RNA, a region is able to fold back (make a loop) and make a strong structure. This loop will block the elongation process and prevent the RNA polymerase to go further ahead. The loop will block transcription and once the RNA polymerase is arrested by this loop, it all depends on the interaction with DNA and RNA. If this region contains many A-U pairs the DNARNA interaction is very unstable (because A interacts with U with only two hydrogen bonds) and it finally dissociates. This is called an intrinsic terminator. Some other genes in bacteria need the help of a protein called Rho. Rho binds to RNA and moves throw it until it arrives to the RNA polymerase and helps the complex to dissociate. In these cases, the interaction between DNA-RNA is more stable because in this region of the RNA there are no Us. These terminators are called rho-dependent terminators.
o Terminator in eukaryotes: We don’t know a lot but it seems that most of the genes use the intrinsic termination.
• Important differences between DNA and RNA: As DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds. The two principal differences on the structure are: 1. The nucleotides in RNA are ribonucleotides. This means that they contain the sugar ribose rather than deoxyribose. The chemical structure of the ribose contains an oxygen group at C2’ that may help 2014/2015 Molecular biology Ingrid Guarro Marzoa breaking the phosphodiester bond, and thus makes RNA less stable than DNA.
2. RNA contains the base uracil instead of thymine. Uracil, as thymine, can base-pair by hydrogen-bonds with adenine and also with guanine.
Although these differences are slight, the structure of both DNA and RNA differ a lot in overall structure: 1. RNA doesn’t have a complementary strand and makes intramolecular paring with itself. RNA is single stranded and can therefore fold up into a particular shape, just as a polypeptide chain. This ability to fold up into complex three-dimensional shapes allows some RNA molecules to have precise structural and catalytic functions.
2. As the oxygen groups on the ribose need to be stabilized, they interact with water. There is some water in RNA.
• Kinds of RNA: mRNA: brings the protein code tRNA: important for translation rRNA: important for translation.
hnRNA: precursor of mRNA snRNA: important for processing hnRNA into mRNA.