Notes for lecture on “Basics of biochemistry and metabolism”

Lecture series D4

“Transcription and translation”

notes based on Alberts et al 4^th ed. (2002) Chapter 6

prepared by T. J. Newman, October 18-October 26, 2005

revised, October 19, 2006

this document not for public use – all images copyright Garland Science Publishing 2002

INTRODUCTION

· In the next few lectures we discuss the mechanisms by which information is transformed to function within the cell

· We will study

o transcription of DNA to mRNA

o translation of RNA to proteins

o control of gene expression

FROM DNA TO RNA

· DNA is not directly coded into proteins

· Instead, a gene is transcribed into a single stranded RNA molecule

· This RNA strand is then modified and translated into a protein by a ribosome

· This process is used by all organisms – it is therefore termed “the central dogma of molecular biology”

· The details of this process differ significantly in prokaryotes and eukaryotes

· Cells can regulate how much of a given protein they produce by

o modulating how many RNA molecules are produced from the corresponding gene

o modulating how many proteins are translated from a given RNA molecule

RNA TRANSCRIPTS

· A gene is transcribed to a single strand RNA molecule (transcript)

o recall, RNA is a nucleic acid, with ribose as the sugar unit in the nucleotide

o the bases in RNA are adenine (A), cytosine (C), guanine (G), and uracil (U)

§ uracil is similar to thymine but lacks a methyl group

o base-pairing in RNA is similar to DNA, although less rigid (e.g. U prefers to bond with A, but will also pair with G)

o because the RNA is in a single strand it can fold up into a three-dimensional structure through base-pairing with itself

§ this allows some RNA molecules to have catalytic activity (these are ribozymes)

· There are several types of RNA produced by transcription

o mRNA – messenger RNA – most genes code for mRNA, which is used for translation into proteins

o rRNA – ribosomal RNA – forms the basic structure of ribosomes

o tRNA – transfer RNA – act as “adaptors” between amino acids and mRNA during protein synthesis

o snRNA – small nuclear RNA – functional in the nucleus, e.g. splicing pre-mRNA

o several other types with specific cell biological functions

· RNA makes up a few per cent of a cell’s mass (excluding water)

o most of this is rRNA, only a few per cent of RNA is mRNA

· DNA transcription shares features with DNA replication

o the double-stranded DNA is unwound at the appropriate place along the sequence

o one of the strands is then used as a template

o (ribo) nucleoside triphosphate molecules are catalytically added to the growing RNA strand

o this strand is bonded to the DNA template strand only over a short region

o the growing tail of the RNA is detached from the DNA template strand, which is then rebound to form DNA double helix

o The enzyme responsible for this is called an RNA polymerase

§ this works along the 5’-3’ direction just like DNA polymerase

· Since the RNA transcript detaches from the DNA double helix, it is possible for several RNA polymerases to work on one gene at the same time (see picture below)

o in eukaryotes, about 20 bases per second are processed

o a typical RNA template will be a few thousand bases in length

o with many polymerases at work, thousands of RNA molecules per hour can be produced from a single gene

· The error rate for RNA template production is far less critical biologically, since the RNA transcript does not encode information in the long-term

o in fact, the error rate of RNA polymerase is about 1000 times greater than that of DNA polymerase

o related to this fact, RNA polymerases can start copying an RNA template without a primer

§ recall that DNA polymerase requires an RNA primer because of its error-correction mechanism

RNA POLYMERASE IN BACTERIA

· RNA transcript production in bacteria is far simpler than in eukaryotes

· The RNA polymerase becomes weakly attached to the DNA and slides along a length until detaching again, unless…

o a subunit (called the s-factor) detects a specific sequence on the DNA

o this sequence is called a promoter

o then the polymerase opens up the double helix and begins to form complementary base-pairing (of RNA bases) with one of the DNA strands

o after about 10 nucleotides, the s-factor detaches and the polymerase elongates and proceeds along the DNA, transcribing RNA (at about 50 bases/second)

o a “rudder” subunit in the polymerase ensures that the RNA transcript is unbound from the single strand allowing the DNA to reform as a double helix after the promoter has passed through a given region

o on encountering another particular sequence (the terminator) the polymerase detaches, along with the transcript

· Termination is thought to work by the terminator coding for a region of RNA which folds into a hairpin structure, which then leverages the RNA polymerase off the DNA strand

· Biologists are not able to efficiently identify promoter sequences

o there appear to be many different promoter sequences, each with its own binding strength for the polymerase

o this indicates, perhaps, that genes coding for heavily-used proteins have promoters which bind strongly, and v.v.

RNA POLYMERASE IN EUCARYOTES

· Eucaryotes have three different types of RNA polymerase (named I, II, and III)

· RNA polymerase II produces all pre-mRNA transcripts

· The two main differences between eucaryotic RNA polymerase and its bacterial counterpart are:

o RNA polymerase in eukaryotes requires a large set of proteins (called general transcription factors) to assemble at the promoter before transcription can begin

o transcription must take place in the highly packed eucaryotic nucleosome/chromatin structures

· The details of transcription factors are complicated and not completely worked out

o in brief, transcription factor proteins help the RNA polymerase to

§ bind to the promoter

§ open up the double-stranded DNA

§ switch RNA polymerase into the “elongation mode”

o once transcription is underway, most of the transcription factors detach from the polymerase

o in vivo, a host of other proteins are required to enable transcription:

§ transcriptional activators bind to specific sequences and aid attachment of polymerase

· this is necessary due to the chromatin structure of the DNA

§ transcriptional mediators interface the activators to the transcription factors

§ chromatin-modifying enzymes aid transcription by opening up the chromatin structure

o in all, over 100 protein subunits (!) must assemble at the promoter site to enable transcription to proceed

· Supercoiling is a problem in transcription for both prokaryotic and eucaryotic cells

o this is relieved in eukaryotes by topoisomerases, just as in DNA replication

o in prokaryotes, negative supercoils are pumped into the DNA by a protein called DNA-gyrase, thus negating the supercoiling due to transcription

· Finally, it is interesting to note that in eukaryotes, an RNA transcript corresponds to a single gene, while in procayrotes, a single transcript can be created from a string of several contiguous genes.

POST-TRANSCRIPTIONAL MODIFICATION

· In prokaryotes, once the RNA transcript has been produced it is ready to interact with the ribosome as an mRNA molecule

· In eukaryotes the situation is very different:

o the transcript (assuming it is destined to be translated into a protein) is called a pre-mRNA transcript

o before it leaves the nucleus, it is heavily modified by enzymes and ribozyme complexes, in particular,

§ the 5’ end is “capped” (with a modified guanine nucleotide)

§ the 3’ end is “polyadenylated” (a complex process resulting in about 200 A’s being added to the mRNA tail)

§ and the introns are removed by a process known as “splicing”

o these processes actually occur as the pre-mRNA transcript is emerging from the RNA polymerase

o the modified ends of the RNA are thought to be used as a check that the template is complete

o the splicing procedure actually enables higher eukaryotes to create different proteins from the same pre-mRNA transcript