Molecular mechanisms of replication, transcription and translation

1. Molecular mechanisms of replication, transcription and translation

2. Procaryotic cell Eucaryotic cell Nucleus separated by membranous envelope from other parts of the cell Lack of nucleus Genetic material (DNA) in circular form called NUCLEOID and no separate compartments for it

3. EUCARYOTIC GENE (is divided) Cis regulatory elements Promoter (AT-rich cassettes) E1 I1 E2 I2 En Ex Ix UAA STOP ATG START UAG UGA Trans regulatory elemets CODING SEQUENCES [EXONS] Non coding sequences [INTRONS] PROCARYOTIC GENE (is continous) GENE 1 E1 E1 Promoter E1 ATG STOP GENE 2 GENE 4 E1 GENE 3 Aleksander L. Sieroń

4. GENE • is a DNA fragment, • Composed of • following sequences: • regulatory • promoter • „structural” Aleksander L. Sieroń

5. EACH GENE HAS AT LEAST TWO VARIANTS, CALLED ALLELES Aleksander L. Sieroń

7. DNA is double stranded RNA is single stranded

8. Para zasad Szkielet cukrowy http://www.genome.gov/Pages/Hyperion//DIR/VIP/Glossary/Illustration/rna.shtml

9. http://www.cytochemistry.net/Cell-biology/ribosome.htm

10. Synthesis of DNA = replication Semiconservative Model http://cropandsoil.oregonstate.edu/classes/css43003/notes4.htm From Cambpell at al.. 1994

11. http://cropandsoil.oregonstate.edu/classes/css43003/notes4.htmhttp://cropandsoil.oregonstate.edu/classes/css43003/notes4.htm

12. 1) INITIATION – replication forks Helicase - separates double helix strands, after removal of super-entangled by topoisomerase cut. Helicase requires energy for its operation, in the form of ATP, to separate the two strands of the helix because they are held together through a number of hydrogen bonds. Topoisomerase - is responsible for the initiation of DNA untangling.The tension at the helix structure in the form of tangled (coiled) and supercoiled structure can break through excessive "stretch" single-stranded DNA, such as the excessive twisting around each other two springs held on the ends. Section of only one of the springs reduces tension caused by twisting and bothspring reels with the participation of topoisomerase Old strand New strand RNA primer Arrows indicate direction of DNA replication. Lagging strand Leading strand Okazaki fragment Topoisomerase Helicase Replication complex ORI Sites of replication origin Aleksander L. Sieroń

13. Aleksander L. Sieroń (2005/2006) Elongation DNA polymerase - progresses along a single strand of DNA recruiting free dNTP ( deoxy - nucleotydo - triphosphate ) to form hydrogen bonds with the appropriate dNTP complementary for them in single strand ( A to T and G to C) , and the formation of covalent phospho-diester bonds from the preceding newly synthetized strands. The energy stored in three phosphates is used to bind each new nucleotide in the growing strand. There are different forms of DNA polymerase, however, the third is the one that is responsible for the progressive synthesis of new DNA strands.DNA polymerase can not start de novo synthesis on a bare thread. It requires primers with 3'OH group to which a dNTP may be attached.DNA polymerase is essentially a complex of several different protein subunits, and is therefore often called holoenzyme.The holoenzyme has the activity of providing control for inserting appropriate base, as well as the properties of nuclease (nucleotide excision) that allow to remove errors occurring during the synthesis. Ligase - catalyzes the formation of phospho-diester bonds between the adjacent 3'OH and 5'fosforanu ends. It eliminates the gap formed after the removal of the RNA primer. DNA polymerase catalyzes the binding of the 5’ end of the primer, but ligase is needed to create a binding site on its 3' end. Single-stranded DNA binding proteins - are important for maintaining the stability of replication forks. Single-stranded DNA is unstable, so these proteins bind to it all the time when it stays as a single strand, protecting it from degradation. Original strand New strand RNA primer Arrows indicate direction of DNA replication. Lagging strand Leading strand Okazaki fragment Primase - is part of a protein complex called primeosome. This enzyme synthesizes a short primer, single-stranded RNA complementary to DNA, which acts as a substitute for the 3'OH end of DNA polymerase synthesis starting a new thread. RNA primer is then removed by RNase H, and the resulting gap is filled by DNA polymerase I. DNA polimerase Replication complex Primase Ligase (after RNAse H) ORI Single-stranded DNA binding proteins

14. Major proteins in DNA replication Initiation of replication Double helix unwinds exposing single stranded templates Helicase, Topoisomerase and proteins binding to single stranded DNA (SSBP) Synthesis of leading strand Synthesis of lagging strand RNA primers annealing Elongation Replacement of RNA primer with DNA PRIMASE DNA POLYMERASE DNA POLYMERASE Annealing of RNA primers for Okazaki fragments Fragments elongation Replacement of RNA primer with DNA Ligation of Okazaki fragments PRIMASE DNA POLYMERASE DNA POLYMERASE LIGASE

15. Initiation of the replication It starts at sites on DNA called "origin" (ori). The sites are characterized by specific sequences serving for: • Binding of initiation proteins, • Separation of the two strands and initiation of replication process, • Binding of replication enhancing proteins.

17. Regulation of replication Although errors in newly synthesized strand of DNA consist only 1 per billion nucleotides, at the beginning of replication their number is 1 per 100 thousands nucleotides.

18. Termination of replication • End of adding of new nucleotides in particular sites, • Connection of new DNA into complete chromosomes, • Segregation of chromosomes to daughter cells.

19. Telomeres

20. DNA stores genetic information in stable form, which can be easily synthesized Expression of genetic information requires of flow of information from DNA to proteins trough RNA

21. transcription translation proteins Elementary dogma in biology Mikro i antisense RNA siRNA Unidirectional linear flow of genetic information reverse transcription Post-transcriptional and post-translational modifications with sequence swapping (lack of corresponding coding sequences in genes) Aleksander L. Sieroń

22. DNA is a genetic material Genetic code is the link between sequence of bases in DNA (or its transcript RNA) and sequence of amino acids in a protein

23. Geneticcodecharacteeristics: • Three nucleotidesencode for one aminoacid. • The codeis not overlapping. • The codelacksspaces, comas and stops. • Geneticcodeisdegenerated. • Geneticcodeisuniversal.

24. GENETIC CODE Codons for DNA Amino acids

25. Geneticcode Second position U C A G U C A G phenyl- alaniane tyrosine cystein U serine STOP STOP leucine tryptophane STOP U C A G histidine C leucine proline arginine glutamine First position Third position U C A G serine aspargine isoleucine A threonine lizyna arginine *metionina U C A G aspargic acid G valine alanine glicyna glutamic acid * i start GGU – glycine CGU - arginine Aleksander L. Sieroń

27. THREE TYPES OF RNA participating in protein bio-synthesis 1. Messenger RNA (mRNA) 2. Ribosomal RNA (rRNA) 3. Transporting RNA (tRNA)

28. RNA All types of cytoplasmic RNA are synthetized in a nucleus from DNA template. RNA is intermediate component ofchromatin.

29. Synthesis of RNA RNA synthesis TRANSCRIPTION Process of transcription of nucleotides DNA sequence into information written in RNA. Synthesis of RNA is catalyzed by large complex enzyme called RNA polymerase. Transcription is conducted in three steps: initiation, elongation and termination.

30. TRANSCRIPTION INITIATION Nonsense strand - noncoding AACTGT ATATTA Aleksander L. Sieroń

31. NUCLEUS „CYTOSOL” packing, methylation, rearrangement, amplifications, heterochromatin, X chromosome inactivation, reorganization of DNA DNA promoters, enhancers, transcription factors, binding proteins, repressors RNA TRANSCRIPT capping, poly-A tail, splicing, alternative splicing FUNCTIONAL RNA

32. Chromatin modifying complexes Chromatin remodeling complexes REGULATION OF GENE EXPRESION DNA is packed in nucleosomes and higher order structure of chromatin Activators of transcription bind regulatory elements Activators recruit chromatin remodeling and modifying complexes http://web.wi.mit.edu/young/pub/activators_initiation.html

33. RNA Polymerase IIholoenzyme TFIID REGULATION OF GENE EXPRESSION Activators recruit components of transcription apparatus Activators can stimulate activity of assembled transcription apparatus http://web.wi.mit.edu/young/pub/activators_initiation.html

34. Promoter E1 I1 E2 I2 En Ex Ix TRANSCRIPTION IN EUCARYOTES heterogenous nuclear RNA hnRNA E1 I1 E2 I2 En Ex Ix bogate STOP ATG zasad NONCODING SEQUENCES [INTRONS] Coding sequences [EXONS] (cut by CPSF73 protein in complex with other proteins) Adenosine (A) in branching point is critical for enzymatic splicing reaction. http://www.web-books.com/MoBio/Free/Ch5A4.htm Aleksander L. Sieroń

35. TRANSCRIPTION IN EUCKARYOTES Spliceosomeconsists of more than 100 proteins and five small nuclear (sn) RNAs (U1, U2, U4, U5 and U6 snRNA).It is responsible for the removal of sequences from the pre-mRNA. In spliceosomessnRNA are responsible for identifying and pairing with sequences of conformity receipt present in pre-mRNA and probably catalyzing this reaction.Critical for interaction snRNA/pre-mRNA is short duplexbetween U2 snRNA and the sequence of conformity in anintron being removed, called BP sequence (BranchPointSequence - BPS). In the first step of folding the enzyme creates BPS 2'-5 'intron lariat branched and releases the exon of the 5' end. In this reaction, the phosphate at the 5 'end is the place for receipt of nucleophylicattack by a 2' hydroxyl conservative adenosine BPS. In a second chemical step the lariatlaced exons and intron are released after the second attack by the newly formed hydroxyl group at the end of exon 5 'phosphate on the 3' splice site. The folding mechanism is complex. FivesnRNAs and proteins highly associated with themisengaged in it. These ribonucleoproteins form a large (60S) complex, called spliceosom. After the two-stage enzymatic reaction intron is removed and the two adjacent exons are joined. http://www.web-books.com/MoBio/Free/Ch5A4.htm

36. TRANSCRIPTION IN EUCARYOTE http://www.web-books.com/MoBio/Free/Ch5A4.htm

37. Promotor E1 I1 E2 I2 En Ex Ix TRANSCRIPTION IN EUCARYOTE heterogenousnucler RNA hnRNA E1 I1 E2 I2 En Ex Ix bogate STOP ATG NONCODING SEQUENCES [INTRONS] CODING SEQUENCES [EXONS] (cut by protein CPSF73 in complex with other proteins) messenger RNA mRNA E1 E2 Ex En Modification of 5’ end addition of a cap „capping” Modification of 3’ end addition of polyA tail „polyadenylation” C E1 E2 Ex En PolyA http://www.web-books.com/MoBio/Free/Ch5A4.htm Aleksander L. Sieroń

38. „Variation” on transcription Different starting sites (numerous promoter sites) alternative splicing of pre-mRNA

39. Different transcription start sitefor example, the gene sxl (sex lethal)Drosophila melanogaster. At the early stage of embryogenesis Sxl protein arises only female embryo (a).? In the late bud female SXL made et ​​earlier stageitmaskssignal to remove intron 2 through RNA splicing, which leads to the formation of other proteins than in male embryo (b). http://www.web-books.com/MoBio/Free/Ch5A4.htm

40. http://www.web-books.com/MoBio/Free/Ch5A4.htm (i) Without splicing, (ii) to (iv) single splicings, (v) and (vi) double splicing. Arrised mRNAs (i), (iv) and (vi) are bicistronic. Star "*" indicates initiating codon site (AUG).

41. NUCLEUS „CYTOSOL” packing, methylation, rearrangement, amplifications, heterochromatin, X chromosome inactivation, reorganization of DNA DNA promoters, enhancers, transcription factors, binding proteins, repressors RNA TRANSCRIPT capping, poly-A tail, splicing, alternative splicing FUNCTIONAL RNA PRE-TRANSLATION masking, degradation, supply TRANSLATION ribosome binding, final product regulation PROTEIN Cutting off, forming, R-groups modifications, phosphorylation ACTIVE PROTEIN INACTIVE PROTEIN blocking, degradation

42. Some types of RNA synthesized by the cell mRNA - messenger RNA ( called messenger RNA) is a copy of the gene. Formed as a photocopy of the gene sequencesince it rules forming is complementary to one strand of DNA and identical to the second strand of DNA. mRNA is a messenger to transfer information contained in the DNA contained in the cell nucleus to the cytoplasm , where ribosomes using it for the synthesis of proteins. tRNA- transporting RNA ( called transfer RNA) a short RNA of a specific secondary structure and tertiary allowing for binding to the amino acid at one end and the other end of the mRNA. Thus acts as an adapter that provides the building blocks of proteins to the correct location encoded by the mRNA. rRNA- ribosomal RNA ( called ribosomal RNA)is the basic structural building component and functional ribosome. Has a sequence complementary to the appropriate areas of the mRNA and, therefore, allows the binding of mRNA to the ribosome at the production site of the protein. snRNA- short nuclear RNAs ( called small nuclear RNA)is involved in the processing of various RNA as they pass from the nucleus to the cytoplasm. Participates in the regulation of gene expression in the nucleolus. siRNAs- short interfering RNA ( called small interferring RNA)participates in the regulation of gene expression at the level of translation by the degradation of mRNA. Aleksander L. Sieroń

44. RNA chains are created de novo and different in: 5’ to 3’ direction In contrast to DNA synthesis of RNA can begin without primer for polymerase. Berg et al.. 2002

45. Transcription: DNA RNA Information in DNA is copied to RNA Information in one gene is copied to RNA. DNA serves as template for RNA synthesis RNA, which carries information is called mRNA mRNA Carries information (genetic instruction) in DNA Carries information to „machinery” synthetizing proteins in a cell.

46. Posttranscriptional processing

47. Translation Flow of information from mRNA to proteins. A cell is interpreting genetic information and building a protein. Information is a mRNA Translator is a tRNA Constructor is a rRNA

48. Classification of RNA according to its function • Information (messenger); mRNA • Functional (transferring, ribosomal and „small” nuclear and cytoplasmic RNA); tRNA; rRNA • Regulatory: • interfering RNA (RNAi): can participate in resistance to pathogens, genome stabilization trough regulation of transposones and in endogenous gene regulation • Small interfering RNA (siRNA) • Small temporary (stRNAs) • microRNA (miRNA) • lncRNA • …………..

49. mRNA is a series of codons tRNAcarries amino acid to ribosome ribosome, rRNA, is the site for protein synthesis

50. tRNAFunction From Cambpellat al.. 1994