DIFFERENT ASPECTS OF GENE REGULATION

Transcription

1 TARTU UNIVERSITY FACULTY OF BILOGY AND GEOGRAPHY DIFFERENT ASPECTS OF GENE REGULATION TARTU 2005 Sten Ilmjärv

2 2 TABLE OF CONTENT TABLE OF CONTENT...2 GLOSSARY...3 INTRODUCTION THE GENE GENE TRANSCRIPTION Transcription initiation Transcription elongation Transcription termination mrna TRANSLATION Translation initiation Translation elongation and termination DNA REPLICATION RNA SPLICING PROMOTERS ENHANCERS, INSULATORS, SILENCERS & TANSCRIPTION FACTOR mirna...18 REFERENCE...19

3 3 GLOSSARY Eukaryotes: Organisms (ranging from yeast to humans) which have nucleated cells. Gene expression: The process by which a gene's coded information is converted into the structures present and operating in the cell. Expressed genes include those that are transcribed into mrna and then translated into protein and those that are transcribed into RNA but not translated into protein (e.g., transfer and ribosomal RNAs). Phenotype 1 : The observable traits or characteristics of an organism, for example hair colour, weight, or the presence or absence of a disease. Phenotypic traits are not necessarily genetic. Prokaryotes: Organisms, namely bacteria and blue green algae, characterized by the lack of a distinct nucleus. snrna 2 : an abundant class of eukaryotic RNA found in the nucleus, usually less than 300 nucleotides long but excluding ribosomal or transfer RNA of this size; most occur as ribonucleoproteins and they appear to play a role in processing of heterogeneous nuclear RNA. srna 3 : A non-coding RNA (ncrna) is any RNA molecule that functions without being translated into a protein. A commonly used synonym is small RNA (srna). Less-frequently used synonyms are non-messenger RNA (nmrna), small non-messenger RNA (snmrna), and functional RNA (frna). The DNA sequence from which a non-coding RNA is transcribed is often called an RNA gene or non-coding RNA gene. trna 4 : Transfer RNA (abbreviated trna) is a small RNA chain (74-93 nucleotides) that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. Protein 5 : A molecule composed of amino acids linked together in a particular order specified by a gene's DNA sequence. Proteins perform a wide variety of functions in the cell; these include serving as enzymes, structural components, or signalling molecules. Anticodon 6 : a set of three trna nucleotides that binds to the codon. Ribosome 7 : A cytoplasmic cellular structure composed of ribonucleic acid and protein that functions in the synthesis of protein. Ribosomes interact with messenger RNA and transfer RNA to join together amino acid units into a polypeptide chain according to the sequence determined by the genetic code. [24] Primer 8 : A primer is a nucleic acid strand (or related molecule) that serves as a starting point for DNA replication. A primer is required because most DNA polymerases (enzymes that catalyze the replication of DNA) cannot begin synthesizing a new DNA strand from scratch, but can only add to an existing strand of nucleotides. [25]

4 4 INTRODUCTION The purpose of this paper is to give a simple overview about biological aspects and features of gene regulation. That includes DNA transcription regulation, RNA translation regulation, mirnas, promoter region specialities and more. There are many ways to manipulate with genes, so therefore the scale is very wide and it differs between different subjects as human, bacteria, viruses and more. For example some viruses do not have DNA as their infinite source of existence but instead they have RNA. And prokaryotes don t have a nucleus, so therefore they do not need to transport their DNA products like mrna out of the nucleus to the cytoplasm. And even more, products of one cell can influence other cells, so overall it is very hard to follow all the directions, possibilities and mechanisms of gene regulation.

5 5 1. THE GENE First of all what is a gene? The widest spread version explains gene as the most fundamental physical and functional unit of heredity. This means that genes make us who we are and what we can do. The gene is a segment of DNA located in a specific site on a chromosome and it operates the formation of an enzyme or other proteins. The fundamental dogma of molecular biology states that DNA produces RNA, which in turn produces proteins. The action of the protein then produces the phenotype. The gene and of course DNA (deoxyribonucleic acid) consists of nuclei acid, which are as we all know A, T, C, and G. But the DNA has something else to it as well. A strand of DNA contains as said genes, areas that control genes and areas that either have no function or a function which we do not know about. In eukaryotic species, very little of DNA actually encodes proteins and therefore the genes may be separated by vast sequences of junk DNA. As well as there are non-coding sequences between different genes, there can be such areas within the gene as well. These areas are called introns, which can actually be many times longer then the gene itself. In the end introns will be cut off and exons, the areas that code the protein, will be joined together. This happens after transcription and before translation with pre-mrna and is called splicing.

6 6 2. GENE TRANSCRIPTION The genetic code of the DNA is made up of three letter nucleic acids which are overall named as codon. In the end every codon is opposed by an amino acid. But one amino acid can be formed by different codons which differ by their nucleic acid sequence. The first action in producing a protein from DNA is called transcription. The genes that are transcribed together are part of a mutual operon. Also the regulative sequences belong to that operon. Transcription is a process through which a DNA sequence is enzymatically copied by RNA polymerase to produce a complementary RNA. This will lead to formation of mrna. The transcription is divided into three stages: initiation, elongation and termination. 2.1 Transcription initiation In the initiation stage the RNA polymerase specifically finds and binds to the DNA promoter region, which enables the gene to be transcribed. There are different kinds of RNA polymerases: Pol I transcribes the rrna, Pol II the mrna and Pol III 5sRNA genes, the snrna genes and all the trna genes. In prokaryotic RNA polymerase the enzyme consists of five polypeptides: two α subunits, β, β and σ subunit. The transcription elongation is carried through by the RNA polymerase apoenzyme (α 2, β, β ). α subunits are responsible for the assembling of the apoenzyme and they can also interact with transcription activators. β subunit has polymerase activity and β binds to the DNA strain. The belonging of σ factor in the RNA polymerase carries out the specific recognition and binding on the promoter area. Due to the difference in σ factors it is possible for the RNA polymerase binding on different promoter sequences. On the elongation the σ is released. The RNA polymerase responsible for the transcription in eukaryotic cell is more complicated and has more subunits to it. In eukaryotic cell, before binding the RNA Pol II, the transcription factors have to bind on the promoter. This is not necessary in prokaryotic cell, where transcription can start without transcription factors. There are several conserved elements on the promoters which bind with RNA Pol II. Closest to

7 7 the transcription initiation sites are TATA-elements, which have the sequence of TATAAAA and it starts somewhere about -33 nucleotides upstream from the transcription starting point. The next conserved area in the promoter is CAAT (CGCCAATCT), which is 80 nucleotides from transcription starting point. The first transcription factor (TF) that binds to promoter is TFIID, which includes a protein that connects with TBP (TATA-binding protein) and several smaller proteins associated with TBP. Then TFIIA and TFIIB associate with the promoter. The fourth TF called TFIIF binds first to the RNA polymerase and then they bind to other proteins which all together makes up transcription initiation complex. The TFIIF is also responsible for the dsdna to detangle, in order to transcribe the DNA. Then after the initiation of transcription comes transcription elongation. 2.2 Transcription elongation In prokaryotic cell elongation is being catalyzed by RNA polymerase, which now is without the sigma factor. The polymerisation speed of RNA is 40 nucleotides a second. In the exact place where the transcription is taking place the DNA and RNA are bind together along three nucleic acids. But the stability of the complex is still related to RNA and DNA binding with polymerase. The elongation in eukaryotic cell is quite similar to previous. 2.3 Transcription termination The last process in transcription is termination. This takes place when RNA polymerase meets with the death codon or the termination signal. Again this is different in eukaryotes and prokaryotes. In prokaryotes the termination is either dependant on Rho protein or not dependant on Rho protein. The last variant terminators carry regions with high level of G:C, which are accompanied by 6 or more pairs of A:T. The explanation for that is quite logical. The single stranded RNA forms a secondary structure, which calls for the polymerisation to end. And if right after the secondary structure there is a long sequence of only U nucleotides, then this helps the dissociation from the transcription complex. This is due to the weak interaction between nucleotide U and A. If the Rho protein is included in the termination process, then the termination sequences are longer. In that case the Rho protein binds with the growing RNA strain and when the termination point comes or polymerization slows down, then the RNA is being dissociated from the complex by the Rho protein. In

8 8 eukaryotes the termination of RNA Pol III work is also Rho dependant or independent. But the termination of Pol I and Pol II is different. RNA Pol I will stop when it gets signal from 18 nucleotide length sequence where the terminator protein has bind. The termination of Pol II is a bit more difficult. The early transcripts 3 ends are cut off. The termination takes part in 1000 to 2000 nucleotide towards the other end. After the cut an enzyme called poly(a) polymerase adds to the 3 end of the RNA molecule a poly(a) tail. The poly(a) tails increase the stability of mrna and they also play a role in transporting the mrna from nucleus to cytoplasm. The 5 cap is modified with adding the guanine nucleotide to the front of the pre-mrna. This modification is critical for recognition and proper attachment of mrna to the ribosome. It may be also important for other essential processes, such as splicing and transport.

9 9 3. mrna TRANSLATION The second part of protein making is translation process. Translation is protein production from mrna. Protein is the unit that is being formed thanks to translation of mrna. Similarly to transcription translation is carried in three different stages: initiation, elongation and termination. 3.1 Translation initiation The translation differs between eukaryotic and prokaryotic cells. Since prokaryotic cells don t have a nucleus, the mrna can be translated at the same time as transcription. In eukaryotic cell this is impossible, since translation is outside of nucleus and the mrna has to be ready for it to go to cytoplasm. The translation is said to be polyribosomal when there is more than one active ribosome. When trna is charged with the amino acid corresponding to its anticodon, it is called aminoacyltrna. Also mrna is always longer then the coding region. To initiate the translation it is necessary for the small ribosomal unit to bind to the start codon on the mrna. This indicates the starting point where mrna starts coding the protein. Most commonly this protein is AUG, but alternative ones are common in prokaryotes. Initiation consists of the reactions wherein the first aminoacyl-transfer RNA and the mrna are bound to the ribosome. The only transfer RNA (trna) capable of initiating translation is a special initiator trna (trna), which carries the amino acid methionine. The first reactions involve the formation of an initiation complex consisting of methionyl-initiator trna bound to a 40S (measured in Svedbergs, in which a higher S value indicates a greater rate of sedimentation and a larger mass) ribosomal subunit. This reaction is catalyzed by the active form of eukaryotic initiation factor 2 (eif2-gtp), which binds the initiator Met-tRNA to the 40S ribosomal subunit. Note that this binding occurs in the absence of mrna. The mrna is added next. First, cap-binding protein eif4e binds to the 7- methylguanosine cap at the 5 end of the message. Without this cap, the binding of mrna to the ribosomal subunit is often not completed (Shatkin, 1976, 1985), and eif4e is critical for the translation to proceed. However, there is less eif4e than the number of messages in the cell, so it is thought that each mrna has to compete for this cap-binding protein (Thach, 1992). Initiation factor 4A then complexes with

10 10 eif4e and positions itself on a helical hairpin loop in the leader sequence of the mrna. The eif4a (stimulated by eif4b and ATP) unwinds the helix. This step can be rate-limiting if the hairpin loop helix is hidden by some other stable secondary structure. The 40S ribosomal subunit then travels down the message until it reaches an AUG codon in the proper context. Kozak (1986) has shown that not just any AUG will do. For the 40S ribosomal subunit to stop and initiate translation, the nucleotides around AUG are also important. Kozak found the "optimum" sequence to be ACCAUGG. The binding of the 40S subunit to the AUG of the message positions the initiator trna over the AUG codon. Only after the mrna has been properly positioned on the small ribosomal subunit can the 60S ("large") ribosomal subunit bind. This completes the initiation reaction. During this process, the GTP on eif2 is hydrolyzed to GDP. For the eif2 to pick up a new initiator trna, it must be regenerated to eif-gtp by eif2b. 3.2 Translation elongation and termination The next step is elongation. Elongation involves the sequential binding of aminoacyltrnas to the ribosome and the formation of peptide bonds between the amino acids as they sequentially relinquish their trna carriers. As amino acids are joined together, the ribosome travels down the message, thereby exposing new codons for trna binding. The termination of protein synthesis takes place when one of the mrna codons UAG, UAA, or UGA is exposed on the ribosome. These nucleotide triplets (called termination codons) are not recognized by trnas and hence do not code for any amino acids. Rather, they are recognized by release factors, which hydrolyze the peptide from the last trna, freeing it from the ribosome. The ribosome separates into its two subunits, and the cycle of translation begins anew. In E. coli two related proteins catalyze termination. They are specific for different sequences. RF-1 (release factor) recognises UAA and UAG; RF-2 recognises UGA and UAA. Mutations in the RF genes reduce the efficiency of termination, as seen by an increased ability to continue protein synthesis past the termination codon.

11 11 4. DNA REPLICATION Another important procedure that involves DNA is DNA replication, where exact DNA is copied. In order for the DNA to replicate it has to be single stranded. In eukaryotes DNA is double stranded, in prokaryotes and viruses it varies. The segregation of DNA strands is catalyzed by DNA helicase. The synthesis is being carried out with DNA polymerase which needs a primer. This is necessary for the polymerase to be intact with the template strand. The enzyme that synthesises a complementary strand from the RNA into DNA is called reverse transcriptase. In the replication of DNA, results will be that one new strand is combined together with one old strand because both of the olds strands serve as template strands during the replication. The initiation of DNA replication is limited by the existence of specific initiation regions called ori-regions. In difference with bacteria in eukaryotes there is a lot of starting sites for replication. They are allocated all over the chromosome appearing about every bp. In S. cerevisiae there have been isolated ARS elements (Autonomously Replicating Sequences). They are about 50bp long A:T rich sequences.

12 12 5. RNA SPLICING Most of eukaryotic genes contain non-coding sequences introns. In order for the formation of translative mrna it is necessary for the splicing of introns and combing of exons. But what is an intron and what is an exon has at that stage not yet been decided. The decision is made during the splicing process. The regulation and selection of splice sites is done by Serine/Arginine-residue proteins. Alternative splicing is of great importance for genetics. This means that the old idea of one DNA sequence coding one polypeptide is no longer correct. External information is needed in order to decide which polypeptide is produced, given a DNA sequence and premrna. It has been proposed that for eukaryotes it was a very efficient step forward since information can be stored much more economically. There are four known modes of alternative splicing: 1) Alternative selection of promoters this is the only method of splicing which can produce an alternative N-terminus domain in proteins. In this case, different sets of promoters can be spliced with certain sets of other exons. 2) Alternative selection of cleavage/polyadenylation sites: this is the only method of splicing which can produce an alternative C-terminus domain in proteins. In this case, different sets of polyadenylation sites can be spliced with the other exons. 3) Exon cassette mode: in this case, certain exons are spliced out to alter the sequence of amino acids in the expressed protein. But questions still arise: does splicing occur in a particular part of the nucleus, what ensures that the ends of each intron are recognized in pairs so that the correct sequence is removed from the RNA? Introns are removed from the nuclear RNAs of higher eukaryotes by a system that recognizes only short consensus sequences conserved at exon-intron boundaries and within the intron. This requires a spliceosome. There are two splicing sites to cut the intron out: one is 5 a.k.a. left a.k.a. donor site and the other is 3 a.k.a. right a.k.a. acceptor site. In the structural protein coding genes the introns have in both ends very short conserved sequences. There is 100% conservation on both sides next to introns. On the left side there is GT and on the right side conserved area is AG. Inwards from these sites there is a less conserved sequences. One of them is TACTAAC element, which is situated 30 nucleotides upstream from the 3 splicing site. The nucleotides are of course different in RNA. There are three ways to cut out introns: 1) in the case of trna precursors the

13 13 cuts are made by endonucleas and exons are bind together. The enzymes involved recognize specifically pre-trna higher rank structures, not specific nucleotide sequences. 2) In some pre-trna molecules the molecule itself catalyzes the splicing. 3) spliceosome is involved.

14 14 6. PROMOTERS A little more about promoters and the sequences they carry. A promoter differs from other DNA sequence whose role is to be transcribed and later translated. The information for promoter function is provided directly by the DNA sequence: its structure is the signal. In comparison the expressed genes gain their meaning when the information is transferred into the form of some other nucleic acid or protein. The key question is that how the RNA polymerase recognises the specific promoter sequence. Does the enzyme have an active site that distinguishes the chemical structure of a particular sequence of bases in the DNA double helix? What are the requirements for that binding? In the bacterial genome the minimum length required by for unique recognition is 12bp. But the length increases with the size of the genome. Any shorter region is likely to occur, just by chance, a sufficient number of additional times to produce false signals. The sequence doesn t have to be contiguous. There can be some separation in the middle. So because of that the combined length of the sequence is shorter, but since the distance of the separation itself provides a part of the signal it doesn t matter. A sequence is called conserved when the essential nucleotide sequence is present in all the promoters. Still, it doesn t have to be conserved in every single position. A consensus sequence is defined by aligning all known examples so as to maximize their homology. For a sequence to be accepted as consensus, each base has to be reasonably predominant at its position. What is actually happening is that there is still a lot of irrelevant sequence in the binding site. But some short stretches within the promoter are conserved, and they are critical for its function. In the bacterial promoter there are four conserved features: The startpoint is usually, 90% of the time, a purine, which is most commonly the central base of CAT. But it s not really an obligatory signal. The next region is recognisable in almost all promoters. The bacterial -10 TATAAT element, also referred as -10 sequence, was the first promoter element to be identified. The consensus can be summarized in the form

15 15 T 80 A 95 T 50 A 60 A 50 T 96, where the numbers referrers the percentage occurrence of the most frequently found base. Another conserved hexamer is centred ~35 upstream of the starting point. Hence the name -35 sequence. The consensus is TTGACA (T 82 T 84 G 78 A 65 C 54 A 45 ). The sequence between those two sequences is unimportant, but the distance is critical in holding the two sites at the appropriate separation for the geometry of RNA. No element/factor combination is an essential component of the promoter, which suggests that initiation by RNA polymerase II may be sponsored in many different ways. A common feature is that inducible transcription factors bind to sequence elements located upstream of the startpoint. In the end sequence components of the promoter are defined operationally by the demand that they must be located close to the startpoint and help the transcription initiation stage. But there is also sequences called enhancers.

16 16 7. ENHANCERS, INSULATORS, SILENCERS & TANSCRIPTION FACTOR Enhancers are sequences which also stimulate initiation but that are located a considerable distance from the startpoint. DNA may be coiled or otherwise rearranged so that transcription factors at the promoter and at the enhancer interact to form a large complex. Enhancers are often used on temporal regulation. The components resemble those of the promoter. However the elements in enhancers are very close together, but in promoters they could have been separated. The elements functions the same but an enhancer does not need to be close to startpoint. Proteins that bound to the enhancer elements also interact with the proteins from promoter elements. The concept that the enhancer is distinct from the promoter reflects two characteristics. The enhancer position relative to promoter can vary substantially and it can function in either orientation. Enhancer can stimulate any promoter placed in its vicinity. Elements analogous to enhancers which are called upstream activator sequences (UAS) are found in yeast. As enhancers they can function in either orientation but cannot function when located downstream from the startpoint. UAS plays a regulator by binding with regulatory proteins that activates the genes downstream. Enhancer works upon the promoter nearest to it. A nearby enhancer is used to increase the efficiency of promoters or if promoters lack specific regulation they become active only when a nearby enhancer is specifically activated. If to remove enhancer from its original place on the DNA and put it somewhere else, then normal transcription can still be sustained as long as the enhancer is somewhere or anywhere on the DNA molecule. How is it possible for the enhancers to work at any distance or on both sides of the startpoint? There are several possibilities: an enhancer could change the overall structure of the template for example, by influencing the density of supercoiling; it could be responsible for locating the template at a particular place within the cell for example, attaching it to the nuclear matrix; an enhancer could provide an entry site, a point at which RNA polymerase associates with chromatin. So in the end enhancers can be seen as containing promoter elements that are grouped closely together, with

17 17 the ability to function at large distances from the startpoint. The essential role for enhancers may be to increase the concentration of transcription factors in the vicinity of promoter. The organisation of DNA must be flexible enough to allow the enhancer and promoter to be closely located. Elements that prevent the passage of activating or inactivating effects are called insulators and have been identified in two circumstances. When an insulator is placed between an enhancer and a promoter, it prevents the enhancer from activating the promoter. This may explain how the action of an enhancer is limited to a particular promoter. When an insulator is placed between an active gene and heterochromatin, it protects the gene against the inactivating effect that spreads from the heterochromatin. Silencers behave like negative enhancers. Each silencer contains an ARS sequence (an origin of replication) The ARS is bound by the ORC (the origin recognition complex) that is involved in initiating replication. Mutations in ORC gene prevent silencing, indicating that ORC protein binding at the silencer is required for silencing. Any protein which is needed for the initiation of transcription but itself doesn t belong to RNA polymerase is called transcription factors. A factor may recognise another factor, or may recognise RNA polymerase, or may be incorporated into an initiation complex only in the presence of several other proteins.

18 18 8. mirna mirna is actually short for micro-rna. It is usually nucleotides long and is thought to regulate the expression of other genes. mirnas are RNA genes therefore they are not translated into protein. mirna is complementary to a part of one or more messenger RNAs, usually at the site of 3 UTR. The binding of the mirna to the mrna inhibits protein translation. In some cases, the formation of the doublestranded RNA through the binding of the mirna triggers the degradation of the mrna transcript, though in other cases it is believed that mrna won t be degraded.

19 19 REFERENCE Internet pages: Books: Genes VII Benjamin Lewin, Oxford University Press, 2000