Replication of DNA in prokaryotes

The synthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination, distinguished both by the reactions taking place and by the enzymes required.

Initiation At least nine different enzymes or proteins participate in the initiation phase of replication.
They open the DNA helix at the origin and establish a pre-priming complex for subsequent reactions. In prokaryotes the replication begins at a particular region on chromosome called replication origin. In E. coli replication origin is called oriC, which consists of 245 bp. The base pairs of oriC are highly conserved among bacterial replication origins. The sequences which play an important role in the replication initiation are two series of short repeats: three repeats of a 13 bp sequence and four repeats of a 9 bp sequence.

The first protein which participates in the initiation process is the DnaA protein. A complex of about twenty DnaA protein molecules binds to the four 9 bp repeats in the origin, then recognizes and successively denatures the DNA in the region of the three 13 bp repeats, which are rich in A=T pairs. This process requires ATP and the bacterial histonelike protein HU. The DnaC protein then loads the DnaB protein onto the unwound region. Two ring shaped hexamers of DnaB, one loaded onto each DNA strand, act as helicases, unwinding the DNA bidirectionally and creating two potential replication forks. Many molecules of SSB bind cooperatively to single stranded DNA, stabilizing the separated strands and preventing renaturation while gyrase relieves the topological stress produced by the DnaB helicase. Elongation The elongation phase of replication includes two distinct but related operations: leading strand synthesis and lagging strand synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. Leading strand synthesis-It is straightforward. In this, the primase (DnaG protein) synthesizes a short RNA primer which is 10 to 60 nucleotides long at the pre-priming complex. The DNA polymerase III binds to this primer and adds dNTPs to this primer. Leading strand synthesis then proceeds continuously, along with the replication fork. Lagging strand synthesis-Lagging strand synthesis is carried out in short DNA segment called Okazaki fragments. During this, the primase synthesizes an RNA primer at intervals. DNA pol III binds to the RNA primer and add dNTPs & DNA synthesis continues until the fragment extends as far as the primer of the previously added Okazaki fragment, a new primer is synthesized near the replication fork to begin the process again.

Termination Termination of the DNA replication occurs at a terminus region called Ter. The Ter consists of multiple copies of a 20 bp sequence. The Ter sequences allow the replication fork to enter but the replication fork cannot exit these sequences. Ter sequences are associated with a protein called Tus (terminus utilization substance). The Tus-Ter complex can arrest a replication fork from only One direction. Only one Tus-Ter complex functions per replication cyclethe complex first encountered by either replication fork. So, when either replication fork encounters a functional Tus-Ter complex, it halts; the other fork halts when it meets the first (arrested) fork. The final few hundred base pairs of DNA between these large protein complexes are then replicated (by an as yet unknown mechanism), completing two topologically interlinked (catenated) circular chromosomes. DNA circles linked in this way are known as catenanes. Separation of the catenated DNA is carried by topoisomerase IV (a type II topoisomerase) resulting in the formation of two functional DNA.

TRANSCRIPTION The expression of the information in a gene normally involves production of an RNA molecule. The transcription can be defined as A process in which genetic information contained in one strand of DNA is used to synthesize a complementary RNA molecule by RNA polymerase. This process occurs in nucleus of eukaryotes. RNA polymerase
It is an enzyme which catalyzes the synthesis of RNA strand by using DNA as template. Thus all RNA polymerases are DNA-dependent RNA polymerase. It was first discovered in 1960s by Samuel Weiss & Jerar Hurwitz independently. It catalyses a reaction in which 3-hydroxyl end of RNA strand attacks nucleophilically the phosphate of incoming ribonucleoside 5triphosphates (NTP) forming a new phosphodiester bond between growing RNA strand & incoming NTP & releases PPi.

All RNA polymerase requires DNA for its activity and they are most active when bound to a double-stranded DNA. The template DNA strand is copied in the 35 direction thus synthesizing the new RNA strand in 53 direction. Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis of new RNA molecule. RNA polymerase require all four ribonucleoside 5_-triphosphates (ATP, GTP, UTP, and CTP) as precursors for the synthesis of RNA. It lacks proof reading 35 exonuclease activity. Bacterial RNA polymerase The DNA dependent RNA polymerase of E. coli is a large complex enzyme with five core subunits 2. The core enzyme has Mr. of 3,90,000. The sixth subunit is a group of polypeptides designated as . subunit binds temporarily to the core subunits & directs the enzyme to specific binding sites on the DNA. These six subunits constitute the RNA polymerase holoenzyme. The RNA polymerase of E. coli exists in several forms depending on the type of subunit. The most common is 70 which has a Mr. of 70,000. subunit helps only in promoter recognition & helps in binding of RNA polymerase to the promoter region of a gene. Thus it helps in initiation of transcription. After initiation it dissociates from the RNA polymerase & is not present during the elongation phase of transcription. SUBUNI T 70 NUMBER OF AMINO ACIDS 329 1342 1407 91 613 RELATIVE MOLECULAR MASS 36,500 151,000 155,000 11,000 70,000 STRUCTURAL GENE rpoA rpoB rpoC rpoZ rpsD

Promoters Initiation of RNA synthesis occurs at a specific site on the DNA template. RNA polymerase binds its initiation sites through base sequence known as promoters, which directs the transcription of adjacent segments of DNA (genes). In E. coli, RNA polymerase binding occurs within a region stretching from about 70 bp before the transcription start site to about 30 bp beyond it. The promoter region thus extends between positions -70 and +30. Analyses and comparisons of the most common class of bacterial promoters (those recognized by an RNA polymerase holoenzyme containing 70) have revealed similarities in two short sequences centered about positions -10 and -35. These sequences are important interaction sites for the 70 subunit. The consensus sequence at the -10 region is (5)TATAAT(3). This region is called Pribnow box. Sine it is discovered by the

scientist David Pribnow in 1975. The consensus sequence at the -35 region is (5)TTGACA(3). A third AT-rich recognition element, called the UP (upstream promoter) element, occurs between positions -40 and -60 in the promoters of certain highly expressed genes. The UP element is bound by subunit of RNA polymerase. The efficiency with which an RNA polymerase binds to a promoter and initiates transcription is determined in large measure by these sequences, the spacing between them, and their distance from the transcription start site. PROKARYOTIC TRANSCRIPTION Initiation Initiation of transcription consists of two important stages, each stage is multistep process. In the first stage, the polymerase binds to the promoter, forming, a closed complex (in which the bound DNA is intact) and an open complex (in which the bound DNA is intact and partially unwound near the -10 sequence). In second stage, transcription initiation occurs. Transcription is initiated within the open promoter complex. RNA polymerase contains two nucleotide binding sites called the initiation site & the elongation site. The initiation site primarily binds Purine triphosphates ATP or GTP and ATP is usually the first nucleotide in the chain. The initiating nucleoside triphosphate binds to the enzyme in open promoter complex & forms hydrogen bond with the complementary DNA base. The elongation site (also known as catalytic site) is then filled with a NTP which is selected by its ability to hydrogen bond with next base in the DNA template strand. The nucleotides are then joined together by phosphodiester bond. The first base is released from the initiation site & initiation is complete. The initiation phase is not yet complete till polymerization of the first 6-10 nucleotides. After this a conformational change occurs in RNA polymerase which causes the dissociation of subunit from the RNA pol. RNA polymerase leaves the promoter sequence (promoter clearance) & enter into elongation stage. Elongation As the RNA polymerase looses subunit, it enters into the elongation phase. The core enzyme moves along the DNA binding a nucleotide triphosphate that can pair with the next DNA base & opening the DNA helix as it move. The helix recloses as synthesis proceeds. The newly synthesized RNA is released from its hydrogen bonds with the DNA as the helix reforms. RNA polymerase keeps a short RNA-DNA hybrid at transcription bubble in which about 12 bases remain hydrogen bonded to complementary base of DNA. Termination In the termination stage, RNA polymerase stops addition of NTP to growing end of RNA & release RNA from DNA template. It can occur by two different methods. i) independent termination ii) dependent termination independent termination Most -independent terminators have two distinguishing features. The first is a region that produces an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure centered 15 to 20 nucleotides before the projected end of the RNA strand. The second feature is a highly conserved string of three A residues in the template strand that are transcribed into U residues near the 3 end of the hairpin. When a polymerase arrives at a termination site with this structure, it pauses. Formation of the hairpin structure in the RNA disrupts several AUU base pairs in the RNA-DNA hybrid segment and may disrupt important interactions between RNA and the RNA polymerase, facilitating dissociation of the transcript. dependent termination The -dependent terminators lack the sequence of repeated A residues in the template strand but usually include a CA-rich sequence called a rut (rho utilization) element. The protein associates with the RNA at specific binding sites and migrates in the 53 direction until it reaches the transcription complex that is paused at a termination site. Here it contributes to release of the RNA transcript. The _ protein has an ATP-dependent RNA-DNA helicase activity that promotes translocation of the protein along the RNA, and ATP is hydrolyzed by protein during the termination process.

 independent termination

EUKARYOTIC RNA POLYMERASE Eukaryotic RNA polymerase is much more complex than bacterial RNA polymerase. Eukaryotes have three RNA polymerases designated I, II & III. All three are distinct complexes but have certain subunits in common. Each polymerase has a specific function & binds to a specific promoter sequence. RNA polymerase I It is also known as Pol I & RNAP A. It is located in the nucleoli. It synthesizes only one type of RNA, a transcript called preribosomal RNA.Preribosomal RNA contains the precursor for the 18S, 5.8S & 28S rRNAs. RNA polymerase II This enzyme is also called as Pol II & RNAP B. It occurs in nucleoplasm. It is the principal enzyme which synthesizes mRNAs & some specialized RNAs. Pol II enzyme recognizes thousands of promoters which can vary greatly in sequence. Many promoters of pol II have a few sequence features in common including TATA box near base pair -30 & an Inr sequence (Initiator) near the RNA start site at +1. RNA polymerase III It is also known as Pol III & RNAP C. Pol III helps in synthesis of tRNAs, the 5S RNA & some other small specialized RNAs (small nuclear & cytosolic RNAs). RNA polymerase III is located in nucleoplasm. MECHANISM OF TRANSCRIPTION IN EUKARYOTES Transcription of eukaryotes begins with binding of transcription factors and RNA polymerase to the promoter sequence. The first step in transcription is formation of closed promoter complex. It starts with binding of TATA Binding Protein (TBP) to the TATA box. TBP is bound in turn by the transcription factors TFIIB. TFIIB also binds to a DNA on either side of the TBP. TFIIA binds to this, which stabilizes the TFIIB-TBP complex on DNA. This complex is next bound by another complex consisting of TFIIF & RNA polymerase II. TFIIF helps to target RNA pol II to its promoter. Finally TFIIE & TFIIH bind to form a closed promoter complex. TFIIH has DNA helicase activity that promotes the unwinding of DNA near the RNA start site. Thus an open promoter complex formation occurs which is ready for initiation. RNA strand initiation & promoter clearance TFIIH has an additional function during the initiation phase. A kinase activity in one of the subunit ofoTFIIH phosphorylates RNA pol II at several places in the Carboxyl Terminal Domain (CTD) of the polymerases largest subunit. This causes a conformational change in the overall complex initiating transcription. During synthesis of the initial 60-70 nucleotides of the RNA, first TFIIE & then TFIIH are released and RNA pol II enters the elongation phase of transcription. TFIIF remains bound to RNA pol II throughout the elongation.
Elongation, Termination, and Release During this stage, the activity of the polymerase is greatly enhanced by proteins called elongation factors. The elongation factors suppress pausing during transcription and also coordinate interactions between protein complexes involved in the posttranscriptional processing of mRNAs. Once the RNA transcript is completed, transcription is terminated. Pol II is dephosphorylated and recycled, ready to initiate another transcript.

Eukaryotic Transcription factors

POST TRANSCRIPTIONAL MODIFICATION Synthesis of 5 Cap Most eukaryotic mRNAs have a 5 cap, a residue of 7-methylguanosine linked to the 5terminal residue of the mRNA through an unusual 5, 5-triphosphate linkage. The 5 cap helps to protect mRNA from ribonucleases. The cap also binds to a specific cap binding complex of proteins and participates in binding of the mRNA to the ribosome to initiate translation.
The 5 cap is formed by condensation of a molecule of GTP with the triphosphate at the 5 end of the transcript. The guanine is subsequently methylated at N-7, and additional methyl groups are often added at the 2 hydroxyls of the first and second nucleotides adjacent to the cap. The methyl groups are derived from S-adenosylmethionine. All these reactions occur very early in transcription.

RNA Splicing
Many genes for polypeptides in eukaryotes are interrupted by noncoding sequences. These noncoding sequences present in mRNA are known as introns. The coding sequences of mRNA are known as exons. The vast majority of genes in vertebrates contain introns; among the few

exceptions are those that encode histones. The occurrence of introns in other eukaryotes varies. Many genes in the yeast Saccharomyces cerevisiae lack introns, although in some other yeast species introns are more common. Introns are also found in a few eubacterial and archaebacterial genes. Introns in DNA are transcribed along with the rest of the gene by RNA polymerases. The introns in the primary RNA transcript are removed by process known as RNA splicing. During RNA splicing the introns are hydrolysed at their both ends and the exons are joined to form a mature, functional RNA. There are four classes of introns. The first two, the group I and group II ntrons, differ in the details of their splicing mechanisms but share one surprising characteristic: they are self-splicingno protein enzymes are involved. RNA splicing is catalyzed by RNA itself. These catalytic RNAs are known as ribozymes. In eukaryotic mRNAs, most exons are less than 1,000 nucleotides long, with many in the 100 to 200 nucleotide size range, encoding stretches of 30 to 60 amino acids within a longer polypeptide. Introns vary in size from 50 to 20,000 nucleotides. Genes of higher eukaryotes, including humans, typically have much more DNA devoted to introns than to exons. Many genes have introns; some genes have dozens of them. Group I introns

Group I introns are found in some nuclear, mitochondrial, and chloroplast genes coding for rRNAs, mRNAs, and tRNAs. In rare case they are found the bacteria also. The group I splicing reaction requires a guanine nucleoside or nucleotide cofactor, but the cofactor is not used as a source of energy; instead, the 3-hydroxyl group of guanosine is used as a nucleophile in the first step of the splicing pathway. 3- hydroxyl group makes a nucleophilic attack on phosphorus of the 5end of the intron. The guanosine 3-hydroxyl group forms a normal 3, 5-phosphodiester bond with the 5 end of the intron. The 3 hydroxyl of the exon that is displaced in this step then acts as a nucleophile in a similar reaction at the 3 end of the intron. The result is precise excision of the intron and ligation of the exons. Group II introns In group II introns the reaction pattern is similar except for the nucleophile in the first step. The nucleophile this case is the 2-hydroxyl group of an A residue within the intron. The 2 hydroxyl group attacks the phosphorus of the 5 end of intron & forms phosphodiester bond with the 5 end of the intron. A branched lariat structure is formed as an ntermediate. The 3 hydroxyl of the exon that is displaced acts as a nucleophile in a similar reaction at the 3 end of the intron to form phosphodiester bond with the 5 end of the next exon. The intron is displaced as a branched lariat.

Spliceosomal introns Most introns are not self-splicing, and these types are not designated with a group number. The third and largest class of introns includes those found in nuclear mRNA primary transcripts. These are called spliceosomal introns, because their removal occurs within and is catalyzed by a large protein complex called a spliceosome. Within the spliceosome, the introns undergo splicing by the same lariat-forming mechanism as the group II introns. The spliceosome is made up of specialized RNA-protein complexes, small nuclear ribonucleoproteins (snRNPs, often pronounced snurps). Each snRNP contains one of a class of eukaryotic RNAs, 100 to 200 nucleotides long, known as small nuclear RNAs (snRNAs). Five snRNAs (U1, U2, U4, U5, and U6) involved in splicing reactions are generally found in abundance in eukaryotic nuclei. The RNAs and proteins in snRNPs are highly conserved in eukaryotes from yeasts to humans. Spliceosomal introns generally have the dinucleotide sequence GU and AG at the 5 and 3 ends, respectively, and these sequences mark the sites where splicing occurs. The U1 snRNA contains a sequence complementary to sequences near the 5 splice site of nuclear mRNA introns, and the U1 snRNP binds to this region in the primary transcript. Addition of the U2, U4, U5, and U6 snRNPs leads to formation of the spliceosome. The snRNPs together contribute five RNAs and about 50 proteins to the spliceosome, a supramolecular assembly nearly as complex as the ribosome. ATP is required for assembly of the spliceosome, but the RNA cleavage-ligation reactions do not seem to require ATP. Some mRNA introns are spliced by a less common type of spliceosome, in which the U1 and U2 snRNPs are replaced by the U11 and U12 snRNPs. Whereas U1- and U2-containing spliceosomes remove introns with (5)GU and AG(3) terminal sequences, as shown in, the U11and U12-containing spliceosomes remove a rare class of introns that have (5)AU and AC(3) terminal sequences to mark the intronic splice sites. The spliceosomes used in

Attachment of poly(A) tail to 3 end of mRNA At their 3 end, most eukaryotic mRNAs have a string of 80 to 250 A residues, making up the poly(A) tail. This tail serves as a binding site for one or more specific proteins. The poly(A) tail and its associated proteins probably help protect mRNA from enzymatic destruction. Many prokaryotic mRNAs also acquire poly(A) tails, but these tails stimulate decay of mRNA rather than protecting it from degradation. The poly(A) tail is added in a multistep process. The transcript is extended beyond the site where the poly(A) tail is to be added, then is cleaved at the poly(A) addition site by an endonuclease component of a large enzyme complex, again associated with the CTD of RNA polymerase II. The mRNA site where cleavage occurs is marked by two sequence elements: the highly conserved sequence (5)AAUAAA(3), 10 to 30 nucleotides on the 5 side (upstream) of the cleavage site, and a less well-defined sequence rich in G and U residues, 20 to 40 nucleotides downstream of the cleavage site. Cleavage generates the free 3_-hydroxyl group that defines the end of the mRNA, to which A residues are immediately added by polyadenylate polymerase, which catalyzes the reaction

where n 80 processing enzyme Posttranscriptionalto 250. This of rRNA does not require a template but does require the cleaved mRNA as a primer. Posttranscriptional processing is not limited to mRNA. Ribosomal RNAs of both prokaryotic and eukaryotic cells are made from longer precursors called preribosomal RNAs, or pre-rRNAs, synthesized by Pol I. In bacteria, 16S, 23S, and 5S rRNAs (and some tRNAs, although most tRNAs are encoded elsewhere) arise from a single 30S RNA precursor of about 6,500 nucleotides. RNA at both ends of the 30S precursor and segments between the rRNAs are removed during processing. The genome of E. coli encodes seven pre-rRNA molecules. All these genes have essentially identical rRNAcoding regions, but they differ in the segments between these regions. The segment between the 16S and 23S rRNA genes generally encodes one or two tRNAs, with different tRNAs arising from different pre-rRNA transcripts. Coding sequences for tRNAs are also found on the 3 side of the 5S rRNA in some precursor transcripts. In eukaryotes, a 45S pre-rRNA transcript is processed in the nucleolus to form the 18S, 28S, and 5.8S rRNAs characteristic of eukaryotic ribosomes. The 5S rRNA of most eukaryotes is made as a completely separate transcript by a different polymerase (Pol III instead of Pol I).

Processing of pre-rRNA transcripts in bacteria

Processing of pre-rRNA transcripts in vertebrates