Transcription is very similar to DNA replication but there are some important differences:

1. RNA is made of ribonucleotides
2. RNA polymerase catalyzes the reaction
3. The synthesized RNA does not remain base-paired to the template DNA strand
4. Less accurate (error rate: 10^-4)

Transcription

The enzyme RNA polymerase opens the DNA strands and synthesizes an RNA complementary to only one of the DNA strands.

Ribosomal RNA and Messenger RNA 

they have two subunits, a large one and a small one

     Messenger RNA carries the genetic code to the ribosomes

 they are strands of RNA that are complementary to the DNA of the gene for the protein to be synthesized

Transfer RNA

       Transfer RNA translates the genetic code from the messenger RNA and brings specific amino acids to the ribosome for protein synthesis

       Each amino acid is recognized by one or more specific tRNA

       tRNA has a tertiary structure that is L-shaped

  one end attaches to the amino acid and the other binds to the mRNA by a 3-base complimentary sequence

RNA Polymerase and The Transcription Cycle

RNA polymerases come in different forms, but share many features

       RNA polymerases performs essentially the same reaction in all cells

       Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III

RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes

RNA Pol I transcribe the large ribosomal RNA precursor gene

RNA Pol III transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes

The subunits of RNA polymerases

Transcription by RNA polymerase proceeds  in a series of steps

      Initiation                        Elongation                             Termination

Initiation

   Promoter: the DNA sequence that initially binds the RNA polymerase

      The structure of promoter-polymerase complex undergoes structural changes to proceed transcription

       DNA at the transcription site unwinds and a “bubble” forms

       Direction of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)

       Elongation

      Once the RNA polymerase has synthesized a short stretch of RNA (~ 10 nt), transcription shifts into the elongation phase.

       This transition requires further conformational change in polymerase that leads it to grip the template more firmly.

       Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain 

Termination

       After the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript.

       In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.

Topic 2

The transcription cycle in bacteria

2-1 Bacterial promoters vary in strength and sequences, but have certain defining features

s        Holoenzyme= sigma factor + core enzyme

In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity?

Promoters recognized by E. coli s factor

       The predominant s factor in E. coli is s⁷⁰.

       Promoter recognized by s⁷⁰ contains two conserved sequences (-35 and –10 regions/elements) separated by a non-specific stretch of 17-19 nt.

       Position +1 is the transcription start site.

      s⁷⁰ promoters contain recognizable –35 and –10 regions, but the sequences are not identical.

The s factor mediates binding of polymerase to the promoter

      The s⁷⁰ factor comprises four regions called s region 1 to s region 4.

       Region 4 recognizes -35 element Region 2 recognizes -10 element

     Region 3 recognizes the extended –10 element

       Change of the promoter DNA

       the opening of the DNA double helix, called “melting”, at positions -11 and +3.

The striking structural change in the polymerase

the b and b’ pincers down tightly on the downstream DNA

Transcription is initiated by RNA polymerase without the need for a primer

Initiation requires:

The initiating NTP (usually an A) is placed in the active site

The initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme

RNA polymerase synthesizes several short RNAs before entering the elongation phase

Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt. 

The elongating polymerase is a processive machine that synthesizes and proofreads RNA

Transcription is terminated by signals within the RNA sequence

Terminators: the sequences that trigger the elongation polymerase to dissociate from the DNA

       Rho-dependent (requires Rho protein)

       Rho-independent, also called intrinsic (内在) terminator

Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs

Rho (r) -dependent terminators

       Have less well-characterized RNA elements, and requires Rho protein for termination

       Rho is a ring-shaped single-stranded RNA binding protein, like SSB

       Rho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysis

  Rho binds to rut (r utilization) RNA sites

       Rho does not bind the translating RNA

       Rho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysis

Topic 3:
transcription in eukaryotes