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DNA and protein synthesis: RNA structure

Introduction

 

DNA encodes a nucleotide sequence that is required for RNA, amino acid and protein production within a cell. RNA has more functions than DNA and there are many different types. DNA and RNA are both comprised of nucleotides, but DNA contains a deoxy-ribose sugar whereas RNA contains ribose, and RNA contains uracil while DNA contains thymine. The first stage in protein synthesis is transcription – where the DNA code for a gene is copied to form a single-stranded mRNA molecule (remember DNA is double stranded). As well as the information required for synthesis of the protein, the mRNA carries non-coding sequences either side of the coding region. These non-coding regions are regulatory sequences that bind the protein production machinery – the ribosome. Translation is the process of generating the protein from the mRNA template. As the mRNA moves through the ribosome (site of protein synthesis) tRNA brings the corresponding amino acid into the ribosome and it is then joined to the growing polypeptide chain. RNA has no regular structure like the double helix of DNA. Instead it is a complex and unique molecule capable of taking on different structures.

 



Structure of RNA

 

The primary structure of RNA is determined by sequence of the nucleotides. Higher order structure of RNA depends on how these bases pair. Nucleotides are paired by hydrogen bonds between complimentary bases and base-pair geometry (correctly paired bases fit into the active site of the polymerase, whereas incorrectly paired bases do not). Watson-Crick base pairing is based on the number of hydrogen bonds the bases can form with each other. Guanine and cytosine form 2 hydrogen bonds together, and adenine and uracil (or thymine in DNA) form 3 hydrogen bonds together. The ribose backbone differs from the deoxyribose backbone as it has an extra OH group which can be involved in hydrogen bonding.

 

In DNA the polynucleotides are double stranded and form a helix with higher order structure largely controlled by proteins such as histones. In RNA however, the strands are single and unpaired which means that short sequences within the strand are free to pair up with partially complimentary regions. For example, a very adenine-rich sequence may bind to a uracil-rich region.

 

Secondary structure of RNA

 

Secondary structure involves interactions between bases to form structures such as helices, hairpin loops, bulges and internal loops as shown in the figure on the right. Bulges and internal loops are formed from mis-matched base pairs. Hairpin loops are formed when two complementary strands form a double helix (the stem) with a loop of mis-matched bases at the end.

 

tRNA is a common example of RNA formed from hairpin loops. All tRNA have the cloverleaf structure at the secondary stage. The acceptor stem is hydrogen bonded, and the arms are formed from hydrogen bonded stems and a single-stranded loop. The double-stranded region forms a short, stacked right-handed helix.

 

Additional structural features of RNA that enable it to form complex secondary structures include "wobble" base pairs, such as the G-U base pairing which occurs in almost all RNA types.

 

RNA secondary structure types

Function of RNA secondary structure

Secondary structure serves a range of purposes in RNA. Partially it stabilises RNA in the cell by hiding the charged groups and is involved in the localization of some RNAs, for example during cell division.

It can also facilitate its interaction with other biomolecules. In the case of the yeast tRNAphe, the cloverleaf secondary structure, form a 3D tertiary compact and highly stable L-shape – enabling the tRNA to fit into the P and A sites of the ribosome. Stacking interactions between the bases in the helical arm stems stabilise the structure further.

RNAs can interact with each other through quaternary structures which are similar in nature to the secondary structures. For example small interfering (si) RNA binds to other types of RNA to prevent it from functioning. For example it can bind to mRNA and block transcription of genes. For this reason it is sometimes known as silencing RNA. This effect is sometimes copied by researchers to prevent expression of target genes.

 

RNA secondary structure prediction

 

The identification and modelling of different nucleic acid structures is vitally important for the design of novel nanotechnology devices and for synthetic biology such as drug design. Structure prediction can begin with analysis of the sequence data and comparing alignment data. From this the secondary structure can be predicted and consequently the 3D structure. There are many computer programs designed specifically for nucleic acid structure prediction. For example, the program M-Fold uses a thermodynamic approach as do many others. This basically means the program assumes the best structure is that with the lowest Gibbs free energy. This program has now been modified to be able to overcome the problem of pseudoknots (a secondary structure formation with multiple stem-loop structures in which half of one stem links to another stem), which are more difficult to predict due to their overlapping nature.

 

Other programs, such as Pfold, use stochastic context-free grammars (SCFG) which predict structure based on probable pairwise correlations from multiple sequence alignments. Programs such as Foldalign and StemLoc use multiple sequence alignment for finding the non-coding RNAs within genomic sequences. 3D structure prediction is more complex than secondary structure prediction and there are fewer methods available for researchers. The program MC-Fold predicts the secondary structure based on non-Watson-Crick base pairing, and then MC-Sym can be used to construct the 3D structure based on an output list of possible structures which can be compared to an available high-resolution RNA structure. The 3D structure can then be used in the design of a novel 3D model, which can be synthesised and assembled and characterised using biophysical methods.

 

RNA binding proteins

 

RNA binding proteins (RBPs) bind either double or single-stranded RNA in the cytoplasm or nucleus. They bind at a specific RNA recognition motif (RRM). RBPs bind to RNA and help regulate translation and post-translational events such as splicing, editing and transport.

 

Sm proteins are a type of RBP and have been extensively studied and their structure is solved. The secondary structure of an Sm protein consists of five beta-sheets and a short helix at the N-terminal. At the tertiary level, these beta sheets are bent to form a barrel shape. To form a RBP, generally seven Sm proteins join together to form a ring, or a torus, which is approximately 7nm in diameter with a 2nm internal hole. snRNA generally binds to the lumen of the ring, with one nucleotide binding per Sm subunit and in turn passing to each subunit before exiting the ring through the other side. snRNA complexes with Sm proteins (in a ring structure) to form snRNPs which complex to form a spliceosome, which is involved in the splicing of introns from pre-mRNA to form mature-mRNA.

 



Definitions:

 

  • rRNA – ribosomal RNA, components of ribosomes 
  • mRNA – messenger RNA, encode an amino acid sequence of a polypeptide from a gene
  • tRNA – transfer RNA, read the mRNA and transfer the amino acids to the growing protein chain
  • snRNA – small nuclear RNA
  • pre-rRNA – preribosomal RNA
  • snoRNA – small nucleolar RNA
  • hnRNA – heterogeneous nuclear RNA
  • scRNA – small cytoplasmic RNA
  • siRNA - small interfering RNA
  • snRNA – small nuclear RNA
  • snRNP – small nuclear ribonucleoprotein

 

References

        1. Nelson, D. L. and Cox, M. M. (2005) ‘Leninger Principles of Biochemistry’ (4th Ed.) W. H. Freeman and Company, New York.
        2. Batey, R. T., Rambo, R. P. and Doudna, J. A. (1999) ‘Tertiary Motifs in RNA Structure and Folding’. Angew. Chem. Int. Ed. 38, 2326-2343
        3. Varani, G. and McClain, W. (2000) The G-U Wobble Base Pair. EMBO Reports. 1: 18-23
        4. Gonzalez, I. et al (1999) ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation. Current Biology. 9: 337-340.
        5. Anderson, E. B. (2010) ‘Prediction and design of DNA and RNA Structures.’ New Biotechnology. 27(3): 184-193
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