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DNA Replication: Mechanisms, Processes, and Key Players, Study notes of Molecular biology

A comprehensive overview of dna replication, a fundamental process in molecular biology. It delves into the history of dna structure discovery, explores different models of replication (theta, rolling circle, linear), and outlines the essential proteins involved in prokaryotic dna replication. The document also highlights the importance of dna replication for cell division and inheritance, emphasizing the semi-conservative nature of the process.

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MBG 2040 Week 8
The central dogma of molecular biology
was articulated by Francis Crick in 1958.
oThe understanding of the processes encapsulated in the central dogma has earned
many nobel prizes
There are 2 main parts to the central dogma:
oThe replication of DNA that is passed onto 2 daughter cells,
oThe processes of transcription and translation that use the information stored in
DNA to make RNA and/or proteins
Some RNAs remain as RNA and always function this way, they are not translated into
proteins. The specialized process pf reverse transcription utilizes an enzyme, reverse
transcriptase, to copy RNA information back into DNA
oThis is an enzyme found in many retroviruses
The Importance of DNA replication
It ensures that an exact copy of the species’ genetic information is passed from cell to cell
during growth and from generation to generation
If DNA failed to replicate itself, the process of mitosis and meiosis would come to a halt
and so it is therefore essential to continue all life
Discovery of DNA structure-History
The 1962 Nobel Prize went to J. Watson, F. Crick, and M. Wilkins for the discovery of
DNA structure. R. Franklin also contributed to this discovery, but she passed away in
1958
Watson and Crick stated: “this structure has novel features which are of considerable
biological interest” and it was ranked #1 of the 15 greatest understatements of all time.
Discovery of DNA structure
DNA is consisted of complementary base pairs (Adenine pairs with Thymine, Guanine
pairs with Cytosine) Purines are A and G, pyramidines are T and C
There are 3 hydrogen bonds (HB) for G-C pairs, and 2 HB for A-T pairs.
The strands run 5’ to 3’ and are antiparallel to each other, which means that they run
opposite directions.
The shape of a DNA strand is called a right-handed double helix or B-DNA; this is the
most common form in living cells
There are 10 base pairs per turn
There are 0.34nm between stacked bases, and 3.4nm per helical turn
DNA replication is semi-conservative
The Watson-Crick double helical model suggested a possible semi-conservative copying
mechanism
The parental DNA molecule separates into 2 parental strands, where they act as templates
for new complementary strands and 2 identical daughter molecules are created
After 1 round of DNA replication, each DNA strand contains 1 parental and 1 newly
synthesized strand
Each of the original parental DNA strands serves as a template to produce a new,
complementary daughter DNA strand.
The possibilities of DNA replication (disproven)
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MBG 2040 Week 8 The central dogma of molecular biology  was articulated by Francis Crick in 1958. o The understanding of the processes encapsulated in the central dogma has earned many nobel prizes  There are 2 main parts to the central dogma: o The replication of DNA that is passed onto 2 daughter cells, o The processes of transcription and translation that use the information stored in DNA to make RNA and/or proteins  Some RNAs remain as RNA and always function this way, they are not translated into proteins. The specialized process pf reverse transcription utilizes an enzyme, reverse transcriptase , to copy RNA information back into DNA o This is an enzyme found in many retroviruses The Importance of DNA replication  It ensures that an exact copy of the species’ genetic information is passed from cell to cell during growth and from generation to generation  If DNA failed to replicate itself, the process of mitosis and meiosis would come to a halt and so it is therefore essential to continue all life Discovery of DNA structure-History  The 1962 Nobel Prize went to J. Watson, F. Crick, and M. Wilkins for the discovery of DNA structure. R. Franklin also contributed to this discovery, but she passed away in 1958  Watson and Crick stated: “this structure has novel features which are of considerable biological interest” and it was ranked #1 of the 15 greatest understatements of all time. Discovery of DNA structure  DNA is consisted of complementary base pairs (Adenine pairs with Thymine, Guanine pairs with Cytosine) Purines are A and G, pyramidines are T and C  There are 3 hydrogen bonds (HB) for G-C pairs, and 2 HB for A-T pairs.  The strands run 5’ to 3’ and are antiparallel to each other, which means that they run opposite directions.  The shape of a DNA strand is called a right-handed double helix or B-DNA; this is the most common form in living cells  There are 10 base pairs per turn  There are 0.34nm between stacked bases, and 3.4nm per helical turn DNA replication is semi-conservative  The Watson-Crick double helical model suggested a possible semi-conservative copying mechanism  The parental DNA molecule separates into 2 parental strands, where they act as templates for new complementary strands and 2 identical daughter molecules are created  After 1 round of DNA replication, each DNA strand contains 1 parental and 1 newly synthesized strand  Each of the original parental DNA strands serves as a template to produce a new, complementary daughter DNA strand. The possibilities of DNA replication (disproven)

 The first possibility was that DNA replicated in a conservative manner, which means that the original DNA helix will be completely preserved in the first round of DNA replication and a brand-new double helix would be generated. o This means that the second round of replication predicts that the original double helix will be preserved and 3 new double helixes will be formed  The second possibility was that DNA replicated in a dispersive manner, where there is no preservation of the original parental double helix but all of the double helixes are hybridized and consist of small segments of both parent DNA and new DNA Semi-Conservative Diagnosis  Meselson and Stahl proved that the semi-conservative model was the correct copying mechanism for DNA  They proved this using a technique called cesium chloride (CsCl equilibrium-density gradient centrifugation to separate double-stranded DNA (dsDNA) molecules of different densities  This permits separation of dsDNA based on density  Heavier DNA sediments further down the CsCl gradient while the lighter DNA migrates toward the top  Newly synthesized DNA strands are labelled with a form of nitrogen (14N) that is lighter than the natural “heavy” form of nitrogen (15N).  When spun in a centrifuge, the densities will separate and it can be seen whether they are new DNA or old DNA  Before any replication takes place, this would show up as one singular band at the bottom of the test tube  After one round of DNA replication, this would show one band of intermediate DNA, because the only strands that are in the mixture are parental strands with a complementary new strand  After the second round of DNA replication, there would be one light band consisting of only new DNA, and one intermediate band that still had all of the parental DNA from before  Any round of replication after this would provide the exact same result with the amount of banding. Theta Replication  This is DNA replication that occurs in most circular DNA o It is bidirectional  It begins from a single origin of replication and proceeds in both directions around the chromosome until 2 newly-synthesized, double-stranded DNA helixes are formed o This type of DNA replication is most commonly used in E. Coli and certain bacterial plasmids  Double stranded DNA unwinds at the replication origin, which produces single-stranded templates for the synthesis of new DNA.  A replication bubble forms, usually with a replication fork at each end  The forks proceed around the circle  Since it happens simultaneously, 2 circular DNA molecules are produced  It is called theta replication because when the forks are preceding it looks like the Greek letter theta Rolling circle replication

 So in double stranded DNA, depending on where the nuclease cuts, the phosphate will reside on the original nucleotide or the adjacent 5’ nucleotide of each chain DNA synthesis is continuous on the leading strand and discontinuous on the lagging strand  For the lagging strand, DNA synthesis begins at the fork and proceeds in the direction opposite that of unwinding, so it soon runs out of template o It starts again at the fork, each time proceeding away from the fork  DNA synthesis on this strand is discontinuous, short fragments of DNA produced by discontinuous synthesis are called okazaki fragments after their discoverer, Reiji Okazaki DNA replication begins from an origin Theta Model  DNA unwinds at the origin  DNA synthesis of the lagging strand proceeds discontinuously in the direction opposite of unwinding  At each fork, DNA synthesis of the leading strand proceeds continuously in the same direction as that of unwinding Linear eukaryotic replication  At each fork, the leading strand is synthesized continuously in the same direction as that of unwinding  The lagging strand is synthesized discontinuously in the direction opposite that of unwinding Rolling-Circle Model  Continuous DNA synthesis begins at the 3’ end of the broken nucleotide strand  As the DNA molecule unwinds, the 5’ end is progressively displaced Proteins involved in Prokaryotic DNA replication  In the bacterium E. coli, the replication origin is called oriC, which occupies a 245 bp segment  It consists of 4 9-mer base pair sequences “TTATNCANA” that bind a protein called DnaA (initiation protein)  It also consists of an “AT”rich region (GATCTNTTNTTT) which unwinds upon DnaA protein binding, which generates a region of single-stranded DNA that permits leading and lagging strand DNA synthesis to begin  Opening of the DNA strands in the AT region promotes the binding of single-stranded binding protein, which keeps the DNA single stranded and linear, and assists in the recruitment of the DNA helicase to each side of the open area to begin further unwinding of the double-stranded DNA  DnaA binds to oriC, causing a short stretch of DNA to unwind, allowing helicase and other single-strand-binding proteins to attach to the single-stranded DNA  DNA helicase unwinds the DNA in the 5’ to 3’ direction, it travels on the lagging strand ahead of the replication machinery  Unwound single stranded DNA is coated with single-strand binding protein to keep DNA single-stranded  DNA helicase binds to the lagging strand template at each replication fork and moves in the 5’ to 3’ direction along this strand, breaking HB and moving the replication fork  Single-strand-binding proteins stabilize the exposed single-stranded DNA

 The unwinding of the helix creates stress ahead of where the unwinding is occurring. If the stress is not relieved the helicase will not be able to continue unwinding the double- stranded DNA  Topoisomerase II (DNA gyrase) proceeds along the DNA ahead of where the helicase is unwinding the DNA and relieves the tension created through “nicking” the DNA strands  This relieves strain ahead of the replication fork to allow it to continue  Helicase-induced unwinding of the double helical DNA causes the DNA ahead of the helicase to be overwound producing positive supercoils that would stop replication o Positive supercoils are where the double-stranded DNA wraps over itself many times  This is when DNA gyrase comes in and nicks the DNA strands ahead of the advancing replication forks to relieve the tension from the positive supercoiling and allowing the replication to continue DNA primase  DNA primase synthesizes a short RNA primer that provides the 3’OH end for DNA polymerase to begin DNA synthesis  Leading strands start first followed by lagging strands  After a section of DNA on both sides of the origin of replication is unwound by the helicases, another enzyme called DNA primase is recruited to the replication fork and synthesizes a short RNA primer  The RNA primer provides the 3’OH end that permits 5’ to 3’ leading strand DNA replication to proceed  As helicase unwinding continues, DNA primase adds primers to begin 5’ to 3’ lagging strand DNA synthesis  The combination of helicase unwinding at each replication fork, adding of RNA primers and leading and lagging strand DNA synthesis permits bidirectional replication to occur away from the origin of replication DNA polymerases  There are 5 DNA polymerases in E. Coli o I, II, III, IV, V o Polymerases I and III are the main two polymerases involved in replication of the circular E. Coli chromosome Activities of DNA polymerases I and III  I aids in removal of RNA primers o It has 5’ to 3’ polymerase and 5’ to 3’ exonuclease activity o Proofreading: has 3’ to 5’ exonuclease activity o Not highly processive, short tract synthesis o Involved mainly in converting Okazaki fragments on the lagging strand to a continuous piece of DNA by removing the RNA primers and replacing it with a short tract of DNA  III is the main replicative polymerase and is highly processive (can copy long stretches of DNA) o This has 5’ to 3’ polymerase activity o Lacks 5’ to 3’ exonuclease activity o Proofreading: has 3’ to 5’ exonuclease activity

Unique aspects of Eukaryotic Chromosome Replication  Shorter RNA primers and Okazaki fragments  DNA replication only occurs during S phase  Multiple polymerases (at least 15) o Pol Epislon: performs leading strand replication o Pol delta: performs lagging strand replication o Pol alpha: synthesizes primase activity  Bidirectional replication from multiple origins of replication on each chromosome  Nucleosomes (two each of histones H2A, H2B, H3, H4 plus H1) need to be removed from parental DNA and properly re-assembled on newly synthesized DNA  Telomeres: Shorten at each round of eukaryotic replication  Eukaryotic cell cycle: o Eukaryotic origins are “prepared” for replication in the G1 phase called “origin licensing”  licensing incolves the assembly and phosphorylatijon of origin specific replication proteins o Actual replication begins in the S phase  The S phasa is the phase within the cell cycle where DNA replication and chromosome duplication occurs from the multiple origins of replication on each chromosome  Disassembly and assembly of nucleosomes is tightly coupled and rapid during DNA synthesis The telomere problem  Chromosome end will be degraded causing chromosome shortening during every round of replication  In circular DNA, replication around the circle provides a 3’OH group in front of the primer; nucleotides can be added to the 3’OH group when the primer is replaced  In linear DNA with multiple origins of replication, elongation of DNA in adjacent replicons provides a 3’OH group for replacement of each primer o The terminal primer is positioned 70-100 nucleotides from the end of the chromosome  In the absence of special mechanisms, DNA replication would leave gaps at the end of the chromosome, and the chromosome would shorten each time the cell divides Telomerase activity extends eukaryotic chromosome ends in replicating cells  To circumvent shortening of eukaryotic chromosomes, eukaryotic cells have an enzyme called “telomerase”.  This has a special RNA component whose sequence is complementary to the TTAGGG repeats found in the telomeres  When the telomerase is bound to the chromosome ends through complementary base pairing, its RNA-templated DNA synthetic activity can extend the length of the chromosome end (in the 5’ to 3’ direction), which normally would get shorted  After extending the length of the chromosome end, telomerase dissociates and then an RNA primer is synthesized, and a complementary DNA strand is synthesized by DNA polymerase  Through the complementary activity of these enzymes, the chromosome end is kept at a normal length

Telomere length and aging  Most human somatic cells have low telomerase activity  Shorter telomeres are associated with cellular senescence and death  Diseases causing premature aging are associated with short telomeres o Progeria o Werner’s syndrome  Result in the inability of cells to maintain the proper length of their chromosomes. This leads to several abnormalities including premature aging  Some cancer cells have high telomerase activity, which is thought to promote their growth Molecular Biology Techniques that are based upon fundamental knowledge of DNA replication  DNA sequencing  Polymerase chain reaction WEEK 8: Transcription and RNA processing Basic Features of RNA  RNA has a ribose sugar (it bears a hydroxyl (-OH) group on its 2’ carbon  RNA contains the base uracil instead of thymine  RNA also has tertiary structure (tRNAs)  RNAs may interact as functional units (quarternary structure) such as in the ribosome The process of Gene expression  In prokaryotes, the coding region of a gene is often a single, continuous unit  Transcription and translation occur at the same time (coupled) Gene expression in Eukaryotes  In eukaryotes, the coding region of a gene is often interrupted: exons are protein coding segments, introns are intervening (non-coding) segments  Transcription and translation are not coupled, but RNA transcripts are made and processed in the nucleus and then must be transported to the cytoplasm for translation Types of RNA  Messenger RNA (mRNA) intermediates that carry genetic information from DNA to the ribosomes (the only RNA translated into protein)  Transfer RNA (tRNA) are adaptors between amino acids and the codons in mRNA  Ribosomal RNAs (rRNAs) are structural and catalytic components of ribosomes  Small nuclear RNAs (snRNAs and snoRNAs) spliceosomes and rRNA, tRNA modification, respectively  Micro RNAs (miRNAs, SiRNA and Crispr RNA) short RNAs that block expression of complementary mRNA’s  Long noncoding RNAs long RNAs that regulate gene transcription RNA  RNA is synthesized in the 5’ to 3’ direction using the 3’ to 5’ DNA template strand o RNA synthesis is complementary and anti-parallel to the DNA template strand  This is only important during transcription because only one of the strands is used as the template o The non-template strand is not usually transcribed

 The “core” RNA polymerase will transcribe any segment of DNA, whereas, specificity for a transcribing from a gene is rendered possible by the inclusion of a specialized subunit called sigma  The core is: α 2 β β’ω ; which will transcribe any DNA sequence  The Holoenzyme is α2 β β’ω σ ; this is the structure of the complete RNA polymerase, which is specific for transcribing genes Functions of the subunits  The alpha subunit (α) is involved in the assembly of the tetrameric core  The beta subunit (β) contains the ribonucleoside triphosphate (rNTP) binding site  The beta-prime subunit (β’) contains DNA template binding region  The omega subunit (ω) helps stabilize the tetrameric core  The sigma subunit (σ) binds to the RNA polymerase tetrameric core and assists in the correct initiation of transcription specifically at the promotor region of the prokaryotic gene o Sigma gives the RNA polymerase specificity for the gene Initiation of transcription  Recognition of the gene promoter region requires the intact RNA polymerase holoenzyme, which consists of the tetrameric core plus sigma factor  Sigma factor recognizes and binds to the -35 element in the promoter region, properly positioning the RNA polymerase to begin transcription  A typical E. Coli promoter region consists of 2 important sequence elements: o The -35 element, where the sigma factor binds o The -10 element, which is due to it’s A/T rich content, is prone to unwinding  The multi-subunit RNA polymerase binds to the promoter region with the aid of the sigma factor o The sigma factor binding with RNA polymerase permits transcription of the gene sequences to begin at the adjacent transcription start site  The -35 sequence is 5’TTGACA 3’  The -10 sequence is 5’ TATAAT 3’  The +1 position is a T or C 5-9 BP after the TATA box Transcription elongation  Elongation occurs when sigma factor is released, and RNA polymerase begins to move along the 3’ to 5’ DNA template strand  A localized region of unwinding called the “transcription bubble” occurs as RNA polymerase moves along the DNA template o The transcription bubble is a localized region of unwinding of approximately 18 BP  Positive supercoils formed in the double-stranded DNA ahead of the advancing RNA polymerase are removed by topoisomerases like in DNA replication  RNA polymerase has both helix unwinding and rewinding activities  The RNA polymerase has built in helicase activity which assists with unwinding the duplex DNA ahead of the polymerase Termination (rho-independent)  Common mechanisms of transcription termination in bacteria

 Weak hydrogen bonding at U:A residues allows mRNA release from DNA when RNA polymerase pauses at terminator  the blocks are “palindromic”: that is, on each DNA strand, the beginning of the sequence on one side is complementary to the beginning of the sequence on the other side. Note also, that at the end of these palindromic sequences, there is a region of A residues on the 3’ to 5’ DNA template strand. The end result of transcribing the 3’ to 5’ DNA template strand is a single strand of RNA that contains these palindromic sequences along with the string of complementary U residues.  As shown, the palindromic sequences can undergo base pairing to form a hydrogen bonded “stem-loop” or hairpin structure in the RNA. During transcription, the hairpin structure resides within the catalytic core of the RNA polymerase and causes it to pause transcription. In so doing, the RNA polymerase and newly-synthesized RNA are released from the DNA template due to weak hydrogen bonding in the adjacent A:U rich region. This ends the process of gene transcription for this particular RNA polymerase. Transcription in eukaryotes  Chromosomes are so large in eukaryotes that regions of active transportation can be visualized microscopically as enlarged open regions of DNA, known as “Balbiani rings”  Most eukaryotes have three RNA polymerases, most eukaryotes only contain RNA polymerases I-III, but plants have 2 additional polymerases o RNA polymerase I: present in all eukaryotes, transcribes large rRNAs o RNA polymerase II: present in all eukaryotes, transcribes Pre-mRNA, some snRNAs, snoRNAs, some miRNAs o RNA polymerase III: present in all eukaryotes, transcribes tRNAs, small rRNAs, some snRNAs, some miRNAs o RNA polymerase IV: only present in plants, transcribes some siRNAs o RNA polymerase V: only present in plants, transcribes RNA molecules taking part in heterochromatin formation  There are specific promoter sequences for genes transcribed by RNA polymerases I, II, or III o Accessory proteins recognize each of these specific types of promoters (through interaction with their DNA sequences) and bind/recruit the appropriate polymerase to begin transcription  Pol. I, pol. II, and pol. III are only recruited to their specific promoters Eukaryotic promoters are more complex than those in prokaryotes  An example is the RNA polymerase II promoter, which consists of a core promoter and a regulatory promoter that aid in positioning transcription proteins and RNA polymerase II to begin transcription  They are characterized by several DNA regulatory elements, which is called the “core promoter” which includes: o The -35 sequence (rich in G/C base pairs): which is the TFIIB recognition element, G/C G/C G/C CGCC o The TATA box (A/T rich) at the -25 position (TATAAAA) o In a region 5’ of the core promoter, some genes also contain additional regulatory regions “regulatory promoter”  Assist in positioning of the RNA polymerase II at the beginning of the gene and facilitating transcription

Termination  Involves cleavage of the pre-mRNA and 5’ to 3’ degradation of the remaining RNA by the Rat1 exonuclease  Transcription terminates when Rat1 reaches RNA polymerase  The long pre-mRNA is cleaved in a special sequence found at the end of the gene by Rat o Rat1 has both endo- and exo-nuclease activities and can both cleave the pre- mRNA and digest the remaining (unusable) part of the transcript o The degradation of the remaining end of the RNA stops transcription by polymerase II causing everything to dissociate, leaving the functional part of the pre-mRNA available for further processing RNA molecules and RNA processing in prokaryotes  In prokaryotes, the coding region of a gene is not interrupted: the sequence of the gene is co-linear with the amino acid sequence of the protein (the mRNA and the polypeptide)  With collinearity, the number of nucleotides in the gene is proportional to the number of amino acids in the protein  The sequence of the messenger RNA corresponds exactly to the sequence of the gene from which it was transcribed  The Shine-Dalgarno sequence: 5’ UAAGGAGGU 3’ is involved in the initiation of translation, and it comes right before the start codon  These regions if the prokaryotic mRNA are all complementary to the DNA of the gene from which transcription occurred  The start codon is ATG, and this part is not transcribed RNA molecules and RNA processing in eukaryotes  Genes are often interrupted: exons are protein segments, introns are intervening (non- coding) segments  The presence of introns in eukaryotic genes was discovered in the 1970s  The removal of introns along with additional RNA processing steps are required to form the mRNA that will be translated into a polypeptide  Transcription of the entire template strand of the gene first produces pre-mRNA, which is then processed to make mature messenger RNA (mRNA) that can be translated into protein  When a gene is transcribed into pre-mRNA, the introns must be removed by a process called RNA splicing  Intron removal makes mRNA, which is a continuous coding segment that can be made into a polypeptide. Transcription generating the pre-mRNA and the various RNA processing steps in eukaryotes all occur in the nucleus Summary of the 3 main processing steps in eukaryotic nuclear pre-mRNA  Addition of 7-methyl Guanosine (7-MG) cap o This is linked to the pre-mRNA by a unique linkage between the 5’ phosphate of the 7-MG and the 5’ phosophate of the first ribonucleotide in the RNA (5’ to 5’ phosphate linkage)  Addition of the polyA tail o Pre-mRNA is cleaved and then a long strip of A residues is added by poly A polymerase  Removal of introns

o Introns in pre-mRNA are removed by a specialized process called “RNA splicing”  The steps of capping and polyadenation serve several functions in the cell-they are important for export of the mRNA, protecting the mRNA from degradation and facilitating translation initiation Further details of the three pre-mRNA processing steps  The 7-methyl Guanosine (7-MG) cap occurs early in the elongation process Addition of the 3’polyA tail  Eukaryotic pre-mRNA is cleaved 11-30nt following the 5’ AAUAAA 3’ sequence in the pre-mRNA  Poly A polymerase adds a string of approximately 200 A residues at the cleaved end Removal of introns from pre-mRNA  Removal of introns must be precise to properly fuse the 3’ end of one exon to the 5’ end of the next exon How does the eukaryotic cell ensure that introns are properly removed?  Every intron has two conserved sequences that are required for its precise removal: o 5’ and 3’ splice sequences containing the junction sequences “GU and AG” respectively o Intron branch point: a conserved “A” residue  These sequences are used by the spliceosome (splicing apparatus) to correctly remove each intron and ligate the 3’ and 5’ ends of each exon Removal of introns  Accomplished by an RNA/protein complex known as the spliceosome o An RNA/protein structure o It contains 5 small nuclear RNAs (snRNAs): designated U1, U2, U4, U5 and U o The snRNAs associate with about 40 small proteins to form small nuclear ribonucleoproteins (snRNP) o snRNPs U1, U2, U4/U6 and U5 assemble to form a complete spliceosome  Splicing involves the ordered assembly of snRNPs to form the splicosome complex, followed by precise cleavage of exon-intron boundaries and ligation.  Part of this unique process involves the formation of a “lariat” structure, which forms when the complete spliceosome forms in step 2 and the 5’ splice site of the intron is cleaved precisely at the G o The intron segment containing the G makes a loop and the 5’ phosphate of the G makes a phosphodiester bond with the 2’OH of the branchpoint A residue in the intron (a unique 5’ to 2’ phosphodiester bond) o After the lariat forms, the 3’ splice site (AG) is cleaved and the intron, along with the spliceosome is excised and the exon ends are ligated  Steps in order:  SnRNP U1 binds to the 5’ splice site and snRNP U2 binds to the branch site  The complete spliceosome assembles and cleaves the transcript at the 5’ splice site  The 5’ end of the intron is joined to the A in the branch site to form a lariat structure, U and U4 are released  The 3’ splice site is cleaved, and the 5’ end of exon 2 is joined to the 3’ end of exon 1. The lariat-shaped intron is released along with snRNPs, U2, U5, and U Recap:

 The anticodon of the tRNA base pairs with the codon of mRNA  The amino acid is covalently attached to the 3’ end of the tRNA  tRNAs often contain modified ribonucleotides through the action of tRNA modifying enzymes and they undergo processing events that remove small introns  they were postulated by Francis Crick in 1956 as adaptors between amino acids and the codons in the mRNAs o Robery Holley shared the 1968 Nobel prize for the nucleotide sequence of a yeast tRNA  tRNAs are the adaptor RNAs used in the translation process to permit codons in mRNA to be read as amino acids by the ribosome  They adopt a type of 3- dimensional structure in solution that favors translation o The 3’ end of the tRNA has a recognition sequence 5’ CCA 3’, which is a feature of all tRNAs and is used as a recognition tag for covalent joining of the amino acid that corresponds to the particular tRNA  The tRNA has an anti-codon sequence that can undergo complementary base pairing with the corresponding codon in the mRNA o Several ribonucleotides in tRNA can be modified by cellular enzymes, which are important for tRNA function and stability Ribosomal RNAs (rRNAs): a key component of the ribosome  Ribosomes are composed of a large and small subunit that are assembled from many different proteins and rRNAs  A ribosome is an RNA machine with key roles in protein synthesis, including the formation of peptide bonds between amino acids  The assembly of prokaryotic and eukaryotic ribosomes is a complex task and involves the participation of several ribosomal proteins and ribosomal RNAs  Prokaryotic rRNAs are made by prokaryotic RNA polymerase, but in eukaryotes, the larger rRNAs are made by RNA polymerase I and the smaller rRNA are made by RNA polymerase III  The 16S rRNA carries the complementary sequence to the Shine-Dalgarno sequence in the mRNA that specifies ribosome assembly  The nucleolus is the site of the eukaryotic rRNA synthesis and ribosome assembly o This is not a nuclear organelle, but just a chromatin dense region in the nucleus  In prokaryotes, there is no nucleus and so rRNA synthesis and ribosome assembly occurs in the cytoplasm Synthesis and processing ribosomal RNAs  Enzymes modify and trim the precursor rRNA transcripts to mature forms in the cell  Prokaryotic and eukaryotic rRNAs are first made through transcription of the rRNA gene producing a long precursor rRNA transcript  The precursor RNA transcript undergoes various endo-and exonucleolytic processing events, as well as some ribonucleotide modifications to form the several mature forms of rRNA used in the assembly of the ribosome  A single tRNA gene evolved within the original rRNA gene that encodes the prokaryotic rRNAs Small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs)  snRNAs act in complexes wuth proteins

 They play roles in post-transcriptional processing of RNA, such as splicing (spliceosome assembly)  snoRNA also act in complexes with proteins  In eukaryotes, they guide the enzymatic chemical modifications of ribosomal RNAs, transfer RNAs and small nuclear RNAs Small micro RNAs (siRNA, miRNA, Crispr RNA)  They are made in both prokaryotic and eukaryotic cells  They have diverse functions in the regulation of gene expression  In eukaryotes: o They act as short (22 nt) single-stranded RNAs that bind to complementary sequences in mRNA o They are produced by cleavage of mRNAs, RNA transposons, and RNA viruses o They regulate and control gene expression in different ways o microRNAs bind to mRNA, forming double-stranded RNA that can either inhibit translation or trigger degradation of the mRNA o siRNAs bind to mRNA to inhibit translation o miRNA binds to mRNA and degrades  In prokaryotic: o Crispr RNA is encoded by DNA sequences found in prokaryotic genomes. o This works in association with the prokaryotic Cas9 nuclease to cleave foreign DNA that might happen to enter a host cell (prevents incorporation of foreign DNA into the host genome-a bacterial defense system) o MicroRNAs constitute a bacterial defense system against the entry of foreign DNA o Crispr RNA can bind to foreign DNA forming a DNA:RNA hybrid that is the target for cleavage by the cas9 nuclease resulting in the eventual destruction of the invading, foreign DNA Long noncoding RNAs  Known to function in eukaryotic cells  Longer than micro-RNAs (200-100000 nt long)  About 80% of the mammalian genome consists of non-protein coding RNAs (very significant)  Their function: o Regulate and control gene expression at the level of transcription or translation by binding mRNA or sequestering microRNAs that control gene expression o Bind and recruit proteins involved in DNA modification (Xist RNA controls the binding of proteins that methylate DNA sequences contributing to X chromosome inactivation in mammals) Week 9: Genetic code Genetic code and translation  Sickle-cell anemia is caused by a single amino acid change (glu to Val) in one of the protein chains that make up hemoglobin in red blood cells Concepts of protein synthesis  The gene is the nucleotide sequence of the DNA  The mRNA transcript is the RNA copy of the template DNA strand of the gene

 Francis Crick and colleagues determined in 1961 that genetic code is indeed a triplet code But what triplets of A, G, C, U in the mRNA specify each amino? Marshall Nirenberg, Gobind Khorana, Philip Leder and colleagues played essential roles in deciphering the Genetic code How?  Marshall Nirenberg and Ghobind Khorana shared the 1968 Nobel prize for this work  One way in which the order of ribonucleotides in the triplet code was solved was by making synthetic homopolymers and adding them to a bacterial cell extract capable of making proteins o By analyzing the polypeptide made from adding the different triplet homopolymers the researchers were able to systematically determine what amino acids were specified by some of the triplet codons as indicated in the slide  Mixed mRNAs (random copolymers) were tested in the same way: they produced polypeptides with different amino acids o Codons for these mixed polypeptides were further verifies by the biochemical approaches  In further experiments, Nirenberg and Leder demonstrated that short mRNAs of known sequence stimulated the binding of ribosomes and the corresponding amino acid bound tRNA o Experiments like these assisted in the identification of the amino acids specified by all mixed codons  The other experiments consisted of the amino acids being specified by biochemical experiments in which a complex of the codon, ribosome and specific tRNA with attached amino acid were recovered on a membrane after filtration o This permitted researchers to determine which tRNA and bound amino acid were specific to each codon The codon table  The genetic code is composed of nucleotide triplets  Of the 4^3 =64 possible triplets, 61 specify amino acids while 3 specify stop codons  The genetic code is degenerate: some amino acids are specified by more than one codon  The genetic code is comma-free becayse each mRNA has consecutive codons that specify amino acids and there are no breaks or pauses  It contains start and stop codons, which are non-sense  It is a nearly universal code Degeneracy in the genetic code  Base pairing between mRNA codons and aminoacyl tRNAs is “anti-parallel” but there is flexibility in binding at the 3rd^ codon position (1st^ anti-codon position) o This is known as the wobble positon  The wobble position permits the tRNA anti-codon, 3’AGG 5’ to bind either the 5’UCC 3’ or 5’UCU 3’ codons in the mRNA o Both codons specify the amino acid, serine  The degeneracy results from a relaxation of the stringency of base pairing at the wobble position  Oftentimes the base in the 3rd^ codon position can be changed and still specify the same amino acid, this feature of the genetic code explains degeneracy Crick’s Wobble hypothesis

 All amino acids except methionine and tryptophan are specified by more than one codon  To account for degeneracy, Crick predicted that several tRNAs must exist for certain amino acids, and that some tRNAs must recognize more than one codon  Stringent base pairing between the codon in mRNA and the anti-codon in tRNA only occurs for the first two bases of the codon  Base-pairing at the third base of the codon is less stringent allowing wobble or flexibility at this position The macromolecules of translation  Ribosomes are made up of polypeptides (>50) and ribosomal RNA (rRNA) molecules (3-

 Amino acid activating enzymes (20)  tRNA molecules (40-60)  soluble proteins (translation factors) involved in polypeptide chain initiation, elongation, and termination Ribosomes  Ribosomes are composed of proteins and several different rRNAs  They are composed of both a large and a small subunit that assemble  A ribosome is an “RNA Machine” with key roles in protein synthesis, including the formation of peptide bonds between amino acids  Ribosome density is indicated in Svedburg units (S) that measure how the ribosomal complex sediments in a gradient during centrifugation  Eukaryotic ribosomes are 80S, and prokaryotic ribosomes are 70S but are composed of 50S (large) and 30S (small) subunits tRNAs  Adapters between amino acids and the codons in mRNA  The anticodon of the tRNA base pairs with the codon of the mRNa  The amino acid is covalently attached to the 3’ end of the tRNA  The normal ribonucleotides in tRNAs (A, G, C, U) are often modified post- transcriptionally by cellular enzymes adding -CH3 or -H  Several tRNAs must exist for certain amino acids and some tRNAs must recognize more than one codon Activation of tRNA by Aminoacyl tRNA synthetase  Cells contain at least one tRNA synthetase for each amino acid o Synthetases catalyze formation of aminoacyl tRNAs  Specific amino acids are joined to their respective tRNAs by an enzyme called “aminoacyl tRNA synthetase” in an ATP-dependent reaction  Special active sites in the enzyme are involved in the covalent attachment of the proper amino acids to the specific tRNAs  The covalent joining of the tRNA and amino acid to make the aminoacyl tRNA (the charged tRNA) requires ATP hydrolysos tRNA charging  An amino acid becomes attached to the appropriate tRNA by the amino-acyl tRNA synthetase in a two-step reaction  In the first step, the amino acid reacts with ATP, producing aminocyl-AMP and PPi o In the second step, the amino acid is transferred to the tRNA and AMP is released  At the end og the tRNA charging, an amino acid is linked to its appropriate tRNA