Chapter 8 – Transcription

Transcription is the term that describes the transfer of genetic information from DNA to RNA, i.e., to copy DNA into RNA

• The process involves three main steps:

• Initiation
• Elongation
• Termination

• RNA Polymerase – this is the enzyme that ‘does’ transcription

• Usually the term ‘transcription’ is used to describe the making of mRNA (i.e., the first step in making a protein) however we could also be talking about making tRNA and rRNA.

• The term also is often thought of as describing the first step in ‘reading’ a gene.

What are the essential chemical characteristics of RNA synthesis?

• You need NTP’s (ribonucleotide triphosphates – ATP, UTP, CTP, GTP)
• Synthesis occurs 5’ à 3’, just like DNA
• The new RNA strand is made off of a template DNA strand; A base-pairs with U and G base-pairs with C.
• In a gene the DNA is double stranded. The RNA will only be made off of one strand (see fig 8-2).
• No primer is needed! The first nucleotide of the new RNA will keep its 5’-triphosphate.

RNA polymerase is a large molecule of about 500 kDa MW

• The holoenzyme is composed of:
• The core -- b (150 kD), b’ (160 kD) and two a (40 kD) subunits
• The sigma factor or the sigma subunit,(s), – there are several varieties of this subunit – typically 70 kD
• The s-factor is responsible for recognizing the DNA site to be transcribed. After it binds and the polymerase starts moving downstream, the s-factor detaches from the core.

The three-dimensional structure of the RNA polymerase holoenzyme was determined by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam in Roger Kornberg lab (Stanford University) by Darst et al. in 1989 ( Nature 340, pp.730-732) at ~ 28 Å resolution. The thumb in the holoenzyme structure defines a deep open groove on the surface of the molecule. The irregularly shaped holoenzyme molecule is approximately 100 x 100 x 160 Å in size.

The Promotor

· This is the RNA polymerase binding site. This is what is recognized by the s-subunit of the RNA polymerase holoenzyme.

· The –10 and –35 consensus regions are the actual s-factor recognition sites. (Fig-8-4 and 8-5). Note that the sequences shown in fig 8-5 are on the non-coding strand.

· The binding of the s-factor to the promotor can be improved if a gene activator protein binds to an enhancer sequence upstream from the promotor. (The catabolite activator protein, CAP, of the lactose operon.

· After RNA polymerase binding, a portion of the double-stranded complex unwinds to form a little bubble. This is called the open promotor complex.

· The RNA polymerase loses its s-factor. NusA (elongation factor) binds to the core and this new complex moves downstream, transcribing the DNA coding strand. See figure 8-7 for overview.

Termination of RNA synthesis

· There are two basic types:

1. Intrinsic Termination – An inverted sequence near the end of the newly synthesized strand of RNA causes the formation of a hairpin loop. This causes a steric problem that makes the RNA synthesizing complex to fall off. Fig 8-8.

2. rho – dependent termination (r-dependent termination). A sequence near the end of the newly synthesized strand binds to the r protein. This protein physically forces the release of the RNA.

The Types or Classes of RNA

· mRNA

o this is the coded information for how to make a particular protein

§ codon = 3 nucleotide code specifying an amino acid

o monocistronic – codes for just one protein

o polycistronic – codes for several proteins

o mRNA code also has start and stop signals for translation

o may have an upstream leader sequence that also regulates activity

o unstable molecule

· rRNA

o stable

o helps to make up the ribosomes

o bacteria have 16s, 23s, and 5s rRNA

o undergoes posttranscriptional modification (RNA processing):

§ pieces are cut from a larger primary transcript

· tRNA

o stable

o carries amino acids to the ribosome for protein synthesis

o has an anti-codon that base pairs with the mRNA codon

o must have a different tRNA for each amino acid

o RNA processing (see fig 8-9):

§ Cut out tRNA from a much longer primary transcript

§ Chemical modification of some bases

Transcription in Eucaryotes:

How is eukaryotic mRNA different from prokaryotic mRNA? The main differences are summarized in fig. 8-10:

· The 5’-end is a methylated guanine – it is said that the mRNA is “capped”

o This methyl guanosine is attached upside down to the first 5’-triphosphate. See fig.9-17 (chapter 9).

· The 3’-end has a poly-A tail added by a special enzyme: poly-A polymerase

· Eucaryotic mRNA is processed (fig 8-11):

o Introns are edited out

o Exons are spliced together

o This is done on an enzyme / snRNP complex called a spliceosome

o Splicing has to be very accurate – missing the splice site by one nucleotide causes a reading frame shift with disastrous consequences.

§ Note the splice site shown on page 160.

What is Southern Blotting – see page 164.