Synthesis of DNA

There is a major difference between DNA polymerase and RNA polymerase: the RNA polymerase can synthesize a new strand whereas the DNA polymerase can only extend an existing strand.  Therefore, to synthesize a DNA molecule, a short RNA molecule (~ 5 - 12 nucleotides) must be synthesize first by a special enzyme.  The initiating RNA molecule is known as a primer, and the enzyme is called primase.

In addition to DNA polymerase and primase, DNA replication requires helicase and single strand binding protein (SSB protein).  The role of helicase is to unwind the duplex DNA.  SSB proteins can bind to both separated strands, preventing them from annealing (reconstitution of double-stranded DNA from single strands).

The replication mechanisms in both bacteria and eukaryotes are similar.  However, eukaryotic DNA polymerases do not contain a subunit similar to the E. coli b subunit.  They use a separate protein called proliferating cell nuclear antigen (PCNA) to clamp the DNA. 

Figure 7-B-2.  Structure of PCNA which is formed by three identical subunits.  PDB ID = 1AXC. 

DNA polymerases can extend nucleic acid strands only in the 5' to 3' direction.  However, in the direction of a growing fork, only one strand is from 5' to 3'.  This strand (the leading strand) can be synthesized continuously.  The other strand (the lagging strand), whose 5' to 3' direction is opposite to the movement of a growing fork, should be synthesized discontinuously. 

Figure 7-B-3.  Steps in the synthesis of the lagging strand.

(a) Comparison between the leading strand and the lagging strand.

(b) The primase first synthesizes a new primer which is about 10 nucleotides in length.  The distance between two primers is about 1000-2000 nucleotides in bacteria, and about 100-200 nucleotides in eukaryotic cells.

(c)  DNA polymerase elongates the new primer in the 5' to 3' direction until it reaches the 5' end of a neighboring primer.  The newly synthesized DNA is called an Okazaki fragment.

(d) In E. coli, DNA polymerase I has the 5' to 3' exonuclease activity, which is used to remove a primer.

(e) DNA ligase joins adjacent Okazaki fragments.

The whole lagging strand is synthesized by repeating steps (b) to (e).

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Mechanism of Reverse Transcription

After the RNA retrovirus enters a host cell, its genomic RNA will be transcribed into a double stranded DNA and then integrated into the host DNA.  The RNA to DNA transcription is calledreverse transcription.  

Figure 4-J-1.  Mechanism of reverse transcription.  The entire process is catalyzed by reverse transcriptase which has both DNA polymerase and RNase H activities.

1. A retrovirus-specific cellular tRNA hybridizes with a complementary region called the primer-binding site (PBS).
2. A DNA segment is extended from tRNA based on the sequence of the retroviral genomic RNA.
3. The viral R and U5 sequences are removed by RNase H.
4. First jump: DNA hybridizes with the remaining R sequence at the 3' end.
5. A DNA strand is extended from the 3' end.
6. Most viral RNA is removed by RNase H.
7. A second DNA strand is extended from the viral RNA.
8. Both tRNA and the remaining viral RNA are removed by RNase H.
9. Second jump: The PBS region of the second strand hybridizes with the PBS region of the first strand.
10. Extension on both DNA strands.  LTR stands for "long terminal repeat".

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Transcription of RNA Genes

The products of RNA genes include rRNA, tRNA and small RNA molecules.  In the mammalian genome, the three rRNA genes, 28S, 18S, and 5.8S, are clustered as a pre-rRNA gene. which is transcribed by RNA polymerase I.  tRNA, 5S rRNA and the U6 snRNA genes are transcribed by RNA polymerase III.  Most snRNA genes are transcribed by RNA polymerase II using the same mechanism as transcribing protein genes.

Transcription by RNA Polymerase I

RNA polymerase I is devoted to the synthesis of pre-rRNA.  Like RNA polymerase II, a pre-initiation complex (PIC) has to be formed before RNA polymerase I can exert its function.  The order of the PIC assembly is illustrated in Figure 4-I-1.

 

Figure 4-I-1.  The PIC assembly for RNA polymerase I.  The regulatory region of pre-rRNA gene contains a core element and an upstream control element (UCE).  Binding of two upstream binding factors (UBF) to both elements may induce DNA looping, and subsequently recruiting TATA-binding protein (TBP) and TBP-associated factors (TAF_I).  Finally, RNA polymerase I joins the complex and completes the assembly process.

Transcription by RNA Polymerase III

Transcription of tRNA gene

The regulatory region of tRNA gene contains A box and B box located inside the transcription unit.  The PIC assembly begins with the binding of TFIIIC to both elements (Figure 4-I-2).

Figure 4-I-2.  The PIC assembly for the transcription of tRNA gene. 

Transcription of 5S-rRNA gene

The regulatory region of 5S-rRNA gene contains a C box, also located inside the transcription unit.  The PIC assembly begins with the binding of TFIIIA to the C box (Figure 4-I-3).

Figure 4-I-3.  The PIC assembly for the transcription of 5S-rRNA gene.   Note that TBP is involved in the transcription by all three types of RNA polymerases in eukaryotes.

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Transcription Mechanisms in Prokaryotes

In prokaryotes, binding of the polymerase's s factor to promoter can catalyze unwinding of the DNA double helix.  The most important s factor is Sigma 70, whose structure has been determined by x-ray crystallography.

(a)

      

(b)

Figure 4-D-1.  The structure of Sigma 70 and its DNA binding site.  (a) Structure of Sigma 70, residues 114 to 448.  PDB ID = 1SIG.  (b) A model for the binding between Sigma 70 and the promoter, based on biochemical studies.  Residues Y425, Y430, W433 and W434 are directly involved in the unwinding (melting) of the double helix.

Note that the promoter is rich in A and T.  The AT pair involves two hydrogen bonds whereas the CG pair involves three hydrogen bonds.  Therefore, AT pairs are easier to separate.  The DNA replication origin is also rich in A and T. 

After the DNA strands are separated at the promoter region, the core polymerase (aabb') can then start to synthesize RNA based on the sequence of the DNA template strand (see .  Since the role of the s factor is mainly to initiate transcription, it will be released after about 10 ribonucleotides have been polymerized.

Elongation of the RNA strand continues until the core polymerase reaches the termination site 

Regulation by Transcription Factors

Regulation of the lac operon transcription by
Catabolite Activator Protein (CAP) and lac Repressor

Activation of the glnA transcription by
Nitrogen Regulatory Protein C (NTRC)
(Example of DNA looping)

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Gene's Regulatory Elements

A gene consists of a transcriptional region and a regulatory region. The transcriptional region is the part of DNA to be transcribed into a primary transcript (an RNA molecule complementary to the transcriptional region). The regulatory region can be divided into cis-regulatory (or cis-acting) elements and trans-regulatory (or trans-acting) elements. The cis-regulatory elements are the binding sites of transcription factors which are the proteins that, upon binding with cis-regulatory elements, can affect (either enhance or repress) transcription. The trans-regulatory elements are the DNA sequences that encode transcription factors.

The cis-acting elements may be divided into the following four types:

Promoter
The DNA element where the transcription initiation takes place.

Enhancer
The element that, upon binding with transcription factors, can enhance transcription. The transcription factors that bind to enhancers are called transcriptional activators.

Silencer
The element that, upon binding with transcription factors, can repress transcription. The transcription factors that bind to silencers are called repressors

Response element
The recognition site of certain transcription factors.

Figure 4-C-1.  Gene organization.  The transcription region consists of exons and introns.  The regulatory elements include promoter, response element, enhancer and silencer (not shown). Downstream refers to the direction of transcription and upstream is opposite to the transcription direction.  The numbering of base pairs in the promoter region is as follows.  The number increases along the direction of transcription, with "+1" assigned for the initiation site.   There is no "0" position.  The base pair just upstream of +1 is numbered "-1", not "0".

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Classes of RNA polymerases

E. coli

An E. coli RNA polymerase is composed of five subunits: two a subunits, and one for each b, b', and s subunit.  b (151 kD) and b' (156 kD) are significantly larger than a (37 kD).  Several different forms of s subunits have been identified, with molecular weights ranging from 28 kD to 70 kD.  The s subunit is also known as the s factor.  It plays an important role in recognizing the  transcriptional initiation site, and also possesses the helicase activity to unwind the DNA double helix.  Nucleotide synthesis is carried out by other four subunits, which together are called the core polymerase.  The term "holoenzyme" refers to a complete and fully functional enzyme.  In this case, the holoenzyme includes the core polymerase and the s factor.

Eukaryotes

There are three classes of eukaryotic RNA polymerases:  I, II and III, each comprising two large subunits and 12-15 smaller subunits.  The two large subunits are homologous to the E. coli b and b' subunits.  Two smaller subunits are similar to the E. coli a subunit.  However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli s factor.  Therefore, in eukaryotes, transcriptional initiation should be mediated by other proteins.

RNA polymerase II is involved in the transcription of all protein genes and most snRNA genes.  It is undoubtedly the most important among the three classes of RNA polymerases.  The other two classes transcribe only RNA genes.  RNA polymerase I is located in the nucleolus, transcribing rRNA genes except 5S rRNA.  RNA polymerase III is located outside the nucleolus, transcribing 5S rRNA, tRNA, U6 snRNA and some small RNA genes.

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function of RNA polymerases

Both RNA and DNA polymerases can add nucleotides to an existing strand, extending its length.  However, there is a major difference between the two classes of enzymes:  RNA polymerases can initiate a new strand but DNA polymerases cannot. Therefore, during DNA replication, an oligonucleotide (called primer) should first be synthesized by a different enzyme.

The chemical reaction catalyzed by RNA polymerases is shown in Figure 4-B-2.  The nucleotides used to extend a growing RNA chain are ribonucleoside triphosphates (NTPs).  Two phosphate groups are released as pyrophosphate (PP_i) during the reaction.  Strand growth is always in the 5' to 3' direction.  The first nucleotide at the 5' end retains its triphosphate group (Figure 4-B-3).

Figure 4-B-2.  The chemical reaction catalyzed by RNA polymerases.

Figure 4-B-3.  Simplified presentation for the chain elongation.  The vertical line represents the pentose and the slanting line denotes the phosphodiester bond.  Bases are designated as N₁, N₂, etc.

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